![[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. 293, Issue 2, 343-350, May 2000
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Massachusetts (K.V., L.L.v.M., D.J.G.); Division of Clinical Pharmacology, New England Medical Center Hospital, Boston, Massachusetts (K.V., L.L.v.M., D.J.G.); and Department of Drug Metabolism, Pfizer Central Research, Groton, Connecticut (R.S.O.)
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
The effect of binding of amitriptyline to human liver microsomes and to microsomes from human B-lymphoblastoid cells on the estimation of enzyme kinetic parameters describing N-demethylation to nortriptyline was investigated using a combination of microsomal binding and in vitro enzyme kinetic studies. Quantitative binding in both matrices increased with higher microsomal protein concentrations (free fractions 0.88-0.32 at 100-500 µg protein/ml in human liver microsomes and 0.82-0.26 at 250-1000 µg protein/ml in microsomes from B-lymphoblastoid cells) and was independent of amitriptyline concentration over a concentration range of 0.2 to 200 µM. Addition of heat-inactivated microsomal protein (50-450 µg/ml) to native human liver microsomes (50 µg/ml) reduced the amitriptyline N-demethylation rate in a protein concentration dependent manner. This effect was greater at lower substrate concentrations and was overcome by saturating concentrations of substrate, thereby decreasing the apparent affinities of the high- and low-affinity components of the N-demethylation process, with minimal effect on the net Vmax. Addition of metabolically inactive microsomes from untransfected human lymphoblastoid cells (750 µg/ml) to CYP2C19 (250 µg/ml protein) increased the apparent Km value for amitriptyline N-demethylation by 3.5-fold and increased the uncompetitive substrate inhibition constant (Ks) by 2.2-fold, making substrate inhibition essentially undetectable. A similar effect was seen with CYP3A4, with a 1.8-fold increase in the S50 (substrate concentration at which half-maximal velocity of a Hill enzyme is achieved). Microsomal binding did not alter the Vmax of either CYP isoform to any appreciable extent. These findings emphasize the importance of incorporating microsomal binding in the estimation of enzyme kinetic parameters in vitro and making appropriate corrections for unbound drug concentrations.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Binding
of drug substrates to in vitro incubation matrices results in an
underprediction of in vivo drug clearance from apparent in vitro
intrinsic clearance determined in enzyme kinetic studies. Correction
for the fraction unbound in the in vitro incubation matrix has improved
the prediction of pharmacokinetic clearance estimates in several
studies, indirectly implicating this phenomenon as the cause of
underprediction of intrinsic clearance (Obach, 1996
, 1997, 1999; Obach
et al., 1997; Carlile et al., 1999). However, the impact of nonspecific
binding on the determination of Michaelis constants
(Km values) describing the drug
biotransformation process has not been directly investigated.
In the present study, we evaluated the effect of binding to human liver microsomes and microsomes from a human B-lymphoblastoid cell line (a widely used model for the production of heterologously expressed CYP isoforms) on the estimation of in vitro enzyme kinetic parameters, using the N-demethylation of the tricyclic antidepressant amitriptyline as the model pathway. The results show that amitriptyline is bound to both human liver and B-lymphoblastoid cell microsomes. The extent of binding increases with increasing microsomal protein concentration and is drug concentration-independent over the range of amitriptyline concentrations generally used in vitro. The predominant effect of microsomal binding on enzyme kinetic parameters was a decrease in the apparent affinity for substrate (i.e., an increase in apparent Km or S50), without any appreciable effect on the reaction velocity at saturating substrate concentrations (Vmax), consistent with an underprediction of intrinsic clearance.
| |
Experimental Procedures |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Materials. Amitriptyline, nortriptyline, clomipramine, desipramine, NADP+, (±)-isocitric acid, MgCl2, and isocitrate dehydrogenase were purchased from Sigma Chemical Co. (St. Louis, MO).
Liver samples obtained from the International Institute for the Advancement of Medicine (Exton, PA) or the Liver Tissue Procurement and Distribution service (University of Minnesota, Minneapolis, MN) were from transplant donors with no known liver disease. The tissue was partitioned and kept at
80°C until the time of microsome preparation as described previously (von Moltke et al., 1993, 1994).
One of four livers used in enzyme kinetic studies of amitriptyline N-demethylation was of poor metabolizer phenotype with
respect to CYP2C19. All livers were normal metabolizers with respect to CYP2D6 activity. Enzymatically inactive microsomes were prepared by
heat-inactivation of native liver microsomes in a boiling water bath
for 4 min, with subsequent confirmation of metabolic inactivity at an
amitriptyline concentration of 500 µM.
Microsomes from cDNA-transfected human lymphoblastoid cells expressing
CYP2C19 or CYP3A4, as well as metabolically inactive control microsomes
from untransfected cells, were purchased from Gentest Corporation
(Woburn, MA), aliquoted, and stored at 80°C. Microsomes from
BTI-TN-5B1-4 insect cells (SUPERSOMES) were purchased from Gentest as
well. Microsomal protein concentrations and CYP content were provided
by the manufacturer.
Spectra/Por 2.1 Biotech membranes (molecular weight cutoff, 15,000;
flat width, 4 mm; diameter, 3.8 mm) were purchased from Spectrum
Laboratories (Rancho Dominguez, CA).
Equilibrium Dialysis.
The binding of amitriptyline to human
liver microsomes or microsomes from human lymphoblastoid cells or
insect cells was determined by equilibrium dialysis using a
modification of a previously described procedure (Obach, 1997).
Dialysis tubing was soaked overnight in deionized water at 4°C. The
tubing was rinsed again in deionized water, followed by dialysis buffer
(50 mM KH2PO4, 5 mM
MgCl2, pH 7.4). Amitriptyline in a methanolic
solution was evaporated to dryness in round-bottomed glass tubes, and
spiked microsomal suspensions of desired drug and protein
concentrations were prepared in dialysis buffer. Dialysis tubing was
cut into 5- to 6-inch-long pieces, and one end was sealed with a knot.
Spiked microsomal suspension (approximately 400 µl) was loaded into
the bag, and the other end was sealed with another knot. The bag was
quickly rinsed in buffer and submerged in 7 ml of dialysis buffer in a 15-ml polypropylene centrifuge tube. The membranes were kept wet throughout the procedure. The tubes were capped and placed in a shaking
water bath at 37°C for 6 h. Preliminary time course studies
suggested that equilibrium was attained within 4 h of dialysis.
Retentates were collected using a 1-ml tuberculin syringe. Dialysates
and retentates were frozen at 20°C until analysis.
). One milliliter of dialysate or 0.15 to 0.25 ml of retentate was transferred to round-bottomed glass tubes
containing internal standard (200 ng clomipramine, evaporated to
dryness from a methanolic solution). Drug-free microsomal suspension
(0.15-0.25 ml) or dialysis buffer (1 ml) was added to the dialysate or
retentate tubes, respectively. Buffer and drug-free microsomal
suspension were both added to standard curve tubes containing 0 and
10 to 50,000 ng amitriptyline and internal standard. All tubes
were alkalinized with 1 ml of 0.25 N NaOH and extracted with 3 ml of
hexane:isoamyl alcohol (98:2) by vortex mixing for 45 s. The tubes
were centrifuged, and the organic phase was transferred to another set
of glass tubes, evaporated to dryness, and reconstituted in 200 µl of
HPLC mobile phase. Then, 25 to 125 µl was injected onto a 30-cm × 3.9-mm steel reverse-phase C18 µBondapack column. The mobile phase
was a 60:40 mixture of 50 mM
KH2PO4 and acetonitrile at
a flow rate of 1.8 ml/min, and ultraviolet detection at 214 nm was
used. Calibration curves were linear, and amitriptyline was quantified
by measurement of peak height ratios with reference to the calibration curve.
The fraction unbound in the microsomal suspension was calculated as the
ratio of amitriptyline concentration in the dialysate to that in the
retentate (Obach, 1997), with the latter being equivalent to total drug
concentration (Wright et al., 1996). In studies of microsomal protein
concentration dependence, the spiked drug concentration was 50 µg/ml
(180 µM). Protein concentrations of 50 to 500 µg/ml (human liver
microsomes) or 250 to 1000 µg/ml (human lymphoblastoid cell
microsomes) were used. When drug concentration dependence was
investigated, the microsomal protein concentration was 200 µg/ml
(human liver microsomes) or 250 µg/ml (human lymphoblastoid cell
microsomes), and amitriptyline was spiked at 3 to 1250 µg/ml (resultant total concentrations in the range of 0.2-200 µM). Pooled human liver microsomes (from seven livers) were used in all equilibrium dialysis experiments.
In Vitro Amitriptyline Biotransformation and Enzyme Kinetic
Analyses.
Incubations of amitriptyline with human liver microsomes
and HPLC analysis of nortriptyline in incubates were performed as previously described (Schmider et al., 1995, 1996; Venkatakrishnan et
al., 1998). A 17-point nortriptyline formation curve (0 and 2.5-500
µM amitriptyline) was used to characterize the kinetics of
amitriptyline N-demethylation by four individual human liver microsomal samples at a microsomal protein concentration of 50 µg/ml.
The impact of microsomal binding on reaction rates and enzyme kinetic
parameters was assessed by generating kinetic curves with the addition
of 50, 150, or 450 µg/ml heat-inactivated human liver microsomal protein.
; Venkatakrishnan et al., 1998).
Kinetic curves were also generated with the coaddition of 750 µg/ml
control microsomes from untransfected lymphoblastoid cells to assess
the impact of microsomal binding on the estimation of enzyme kinetic parameters.
The kinetics of amitriptyline N-demethylation by human liver
microsomes or heterologously expressed CYP isoforms were described by
one of the following models relating nortriptyline formation rate
(v) to concentration of the substrate (S), amitriptyline:
A one-enzyme Michaelis-Menten model with uncompetitive substrate
inhibition for lymphoblast-expressed CYP2C19:
|
(1) |
|
(2) |
|
(3) |
| |
Results |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Binding Studies. The effect of microsomal protein concentration on the unbound fraction of amitriptyline is shown in Table 1. There was essentially no binding at a human liver microsomal concentration of 50 µg/ml, and the free fraction progressively decreased as the protein concentration was increased, with an unbound fraction of only 0.32 at a protein concentration of 500 µg/ml. A similar trend was noted for lymphoblastoid cell microsomes, although the extent of binding was lower than that in liver microsomes. Amitriptyline was also bound to insect cell microsomes with a free fraction of nearly 0.7 at a protein concentration of 250 µg/ml, a concentration typically used in incubations with SUPERMIX and SUPERSOME formulations manufactured by Gentest. The extent of human liver microsomal binding of amitriptyline was increased by heat inactivation to a small extent, with the bound fraction being 26% higher at a protein concentration of 200 µg/ml.
|
|
Effect of Nonspecific Microsomal Binding on Human Liver Microsomal
Amitriptyline N-Demethylation.
The effect of
microsomal binding on the kinetics of amitriptyline
N-demethylation was studied in four human liver
microsomal preparations. The active microsomal protein
concentration was fixed at 50 µg/ml, and kinetic curves were
generated with the addition of 0, 50, 150, or 450 µg/ml
heat-inactivated microsomes from the same liver. Averaged data are
shown in Figs. 2 (primary plots) and
3 (Eadie-Hofstee transformations), and a
two-enzyme model (high-affinity Michaelis-Menten component plus
low-affinity Hill component; eq. 3) was used in the determination of
enzyme kinetic parameters. The choice of this model is consistent with previous enzyme kinetic studies of amitriptyline
N-demethylation (Schmider et al., 1996; Ghahramani et al.,
1997; Venkatakrishnan et al., 1998) and with the underlying profile of
the biotransformation pathway (Olesen and Linnet. 1997; Venkatakrishnan
et al., 1998). The apparent Km and
S50 values of the high- and low-affinity
components, respectively, increased with increasing amounts of
heat-inactivated microsomal protein (i.e., with increasing extent of
microsomal binding) without any consistent effect on the estimation of
the Vmax value of the high- and
low-affinity components (Table 2). Unbound Km and
S50 values were calculated from the estimated
apparent values of these parameters and estimated free fractions (1.0, 0.88, 0.63, and 0.32 at total microsomal protein concentrations of 50, 100, 200, and 500 µg/ml, respectively). These unbound
Km and S50
values are similar at all four protein concentrations (Table 2). Figure
4 shows the effect of addition of
heat-inactivated microsomal protein on amitriptyline
N-demethylation rate as a function of substrate
concentration. In all the livers, the fractional decrement in reaction
rate decreased with increasing substrate concentration, with the effect
being maximal at low (2.5-25 µM amitriptyline) substrate
concentrations. The decrement of reaction rate also increased with
increasing protein concentration, consistent with the results of
binding studies. The effects of substrate concentration as well as
microsomal protein concentration on the fraction of control rate were
statistically significant (P < .001, two-way repeated
measures ANOVA).
|
|
|
|
Effect of Microsomal Binding on Amitriptyline N-Demethylation by Lymphoblast-Expressed CYP Isoforms. Lymphoblast-expressed CYP2C19 (250 µg/ml protein) N-demethylated amitriptyline with high affinity and displayed substrate inhibition (eq. 1). Addition of 750 µg/ml of metabolically inactive control microsomes from untransfected cells produced a 3.5-fold increase in the apparent Km and a 2.2-fold increase in the uncompetitive substrate inhibition constant Ks (Table 3 and Fig. 5, A and B), without a significant effect on the Vmax value. Calculation of Km values based on free drug concentrations using estimated free fractions yielded unbound Km estimates of 8.3 and 9.2 µM, without and with addition of inactive control microsomes, respectively.
|
|
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Amitriptyline is bound to human liver microsomes and microsomes from human B-lymphoblastoid cells. This binding results in an overestimation of Km and S50, without affecting the metabolic rate at saturating substrate concentrations.
Microsomal binding of amitriptyline is consistent with its high
lipophilicity and plasma protein binding
mean free fraction in
plasma 0.057 (Schulz et al., 1985). The amitriptyline free fraction was
drug concentration-independent over the range of amitriptyline
concentrations generally used in vitro (Fig. 1, A and B).
Concentration-independent human liver microsomal binding of
propranolol, imipramine (Obach, 1997), and phenytoin (Carlile et al.,
1999) has also been described, although the free fraction of warfarin
in human liver microsomes increased with increasing drug concentration
(Obach, 1997).
At the highest total concentration of amitriptyline studied
(approximately 200 µM), the bound concentrations of amitriptyline were 70 and 20 µM in human liver microsomes (200 µg/ml protein) and
lymphoblast microsomes (250 µg/ml protein), respectively. The molar
concentration of binding sites and the affinity constant for binding
could not be determined due to the essentially linear relationship
between free and bound amitriptyline concentrations over the
concentration range studied. However, because the molar concentration
of binding sites should in principle be greater than or equal to the
bound drug concentrations (Wright et al., 1996), the measured bound
drug concentrations suggest that human liver microsomes and human
lymphoblastoid cell microsomes contain at least 350 and 80 nmol of
binding sites for amitriptyline/mg of microsomal protein, respectively.
These concentrations are far in excess of the average molar
concentration of total CYP in human liver microsomes, 340 pmol/mg
protein (Shimada et al., 1994), and in these lymphoblastoid cell
microsomal preparations. This suggests that the observed binding is in
fact mainly nonspecific and not reflective of specific interactions
with the enzyme active site.
The amitriptyline free fraction progressively decreased with increasing microsomal protein concentration, in both human liver microsomes and microsomes from B-lymphoblastoid cells (Table 1). It is thus likely that microsomal binding may contribute in part to the nonlinear increase in amitriptyline N-demethylation rate with increasing microsomal protein concentrations at protein concentrations of more than 200 µg/ml (data not shown). Even at a relatively low human liver microsomal protein concentration of 200 µg/ml (the upper limit of the linear range), 40% of the drug is bound to the microsomal matrix.
Consistent with the binding studies, addition of heat-inactivated human liver microsomal protein caused a protein concentration-dependent decrement in reaction rate. The effect became pronounced at low amitriptyline concentrations (Fig. 4), resulting in an increase in apparent Km and S50 of the high- and low-affinity components of amitriptyline N-demethylation, without a consistent effect on the net Vmax (Figs. 2 and 3 and Table 2). Although heat-inactivated microsomes bound amitriptyline to a greater extent than native microsomes (Table 1), the binding was still comparable, thereby allowing their use as a source of nonspecific binding sites devoid of functional enzyme. Unbound Km and S50 values were calculated for each total microsomal protein concentration, using the respective apparent Km and S50 values, and amitriptyline free fractions (Table 2). These unbound Km and S50 values are similar at all four protein concentrations. Therefore, correction for binding reduces much of the variability in Km and S50 attributable to microsomal protein concentration.
As with human liver microsomes, addition of control lymphoblast
microsomes to CYP2C19 or CYP3A4 resulted in an increase in apparent
Km or S50,
respectively, without any appreciable effect on the
Vmax (Table 3 and Fig. 5). Unbound
Km values were calculated for CYP2C19
using the apparent Km values and
amitriptyline free fractions. The unbound
Km values determined without and with
addition of inactive microsomes were similar (8.3 and 9.2 µM,
respectively), suggesting that the alteration of apparent
Km is explained by microsomal binding
of amitriptyline. However, the unbound S50 values
for CYP3A4 determined without and with addition of inactive control
microsomes were not identical (69 and 39 µM, respectively). The
reason for this difference in unbound S50 values
determined at two different microsomal protein concentrations is not
clear and may be related to the increased complexity due to cooperative binding. Exogenously added albumin, for example, decreased the unbound
Km of human liver microsomal phenytoin
hydroxylation (Ludden et al., 1997). Thus, addition of exogenous
protein may in fact alter the true affinity of the enzyme for
substrate, affecting the unbound Km
value. However, it is likely that addition of albumin to an in vitro
reaction mixture is not comparable to addition of microsomal protein.
In any case, the estimation of Km
values based on unbound drug concentrations relies on the assumption that the intrinsic affinity of enzyme for unbound substrate is independent of microsomal protein concentration and that free drug
concentrations, rather than total concentrations, are better estimates
of enzyme-available concentrations in vitro. Although the validity of
this assumption remains unclear, correction for microsomal binding
using the free fraction in incubation matrices clearly improves the
prediction of in vivo clearance from in vitro estimates of intrinsic
clearance for drugs that are extensively bound to microsomal matrices
(Obach, 1996, 1997, 1999; Obach et al., 1997; Carlile et al., 1999).
Thus, unbound Km values based on free
drug concentrations rather than apparent
Km values based on total drug
concentrations are expected to be better estimates of the true
Km based on the existing data.
In addition to its effects on apparent Km and S50, microsomal binding also increased the estimated substrate inhibition constant Ks for CYP2C19-mediated amitriptyline N-demethylation, with negligible substrate inhibition in the presence of 750 µg/ml control (inactive) microsomes (Fig. 5A). Reaction rates in the presence of binding protein at 350 and 500 µM concentrations of amitriptyline were higher than control rates, whereas no such increase in rate was seen for CYP3A4 at saturating concentrations of substrate (Fig. 6). Although microsomal binding should in principle affect only the apparent Km or S50 and not alter the rates at saturating substrate concentrations, this may be true only if the kinetics is consistent with monotonic Michaelis-Menten or Hill functions.
Lymphoblast-expressed CYP isoforms are generally used at microsomal
protein concentrations of 250 to 1000 µg/ml in the determination of
enzyme kinetic parameters. These parameters are subsequently used to
predict relative contributions of individual CYP isoforms to the
overall rate of hepatic drug biotransformation in vivo, accounting for
the relative abundance of each CYP isoform in human liver. Due to the
differing turnover numbers of the CYP isoforms that catalyze a given
drug biotransformation pathway and the relatively similar CYP content
(on a per-milligram of protein basis) of the lymphoblast-expressed CYP
preparations, it may be necessary to use different total microsomal
protein concentrations for different isoforms. Thus, low microsomal
protein concentrations (100-250 µg/ml) may be needed to minimize
substrate consumption by CYP isoforms with a high intrinsic clearance,
whereas higher protein concentrations (500-1000 µg/ml) may be
required to identify and kinetically characterize low-affinity and
low-capacity isoforms, due to limits of analytical assay sensitivity.
For substrates extensively bound to microsomes, the
Km or S50 values
determined for the various CYP isoforms may be biased by the microsomal
protein concentration used, resulting in misprediction of the relative contributions of the various CYP isoforms to net intrinsic clearance. For example, nortriptyline is biotransformed via
E-10-hydroxylation, a reaction that is catalyzed in vitro by
both CYP2D6 (high affinity, high capacity) and CYP3A4 (low affinity,
low capacity) (Venkatakrishnan et al., 1999). Kinetic characterization
of CYP3A4 required the use of a microsomal protein concentration of
1000 µg/ml, whereas a protein concentration of 200 µg/ml had to be
used for CYP2D6 to minimize substrate consumption. Given the structural
similarity with amitriptyline, nortriptyline binding to microsomes may
be significant. Thus, the Km value for
CYP3A4 in relation to CYP2D6 may have been overpredicted.
In addition to its effects on in vitro drug biotransformation kinetics,
microsomal binding of inhibitors can theoretically bias the estimation
of in vitro IC50 or
Ki values in chemical inhibition
studies, potentially causing underestimation of inhibitor potency. In
fact, a microsomal protein concentration-dependent increase in apparent
IC50 values of clotrimazole and ketoconazole versus human liver microsomal midazolam 1'-hydroxylation has been described, although this effect was attributed to inhibitor depletion by specific binding to the enzyme (Gibbs et al., 1999).
Although the physicochemical determinants of microsomal binding are not
completely understood, significant binding has been described for
lipophilic basic drugs like desipramine, imipramine, amitriptyline,
chlorpromazine, and propranolol (Obach, 1997, 1999), whereas warfarin
(Obach, 1997), tolbutamide (Carlile et al., 1999; Obach, 1999), and
dextromethorphan (Witherow and Houston, 1999) are not significantly
bound to human liver microsomes.
Microsomal binding should be measured and incorporated in enzyme kinetic analyses so unbiased kinetic parameters can be determined, based on unbound rather than added (total) drug concentrations. This phenomenon may explain in part the laboratory-to-laboratory variation in Km values reported for a given drug biotransformation pathway, which in turn may lead to incorrect predictions of the relative contributions of high- and low-affinity CYP isoforms to the overall metabolic rate, when varying microsomal concentrations of the heterologously expressed CYP isoforms are used in kinetic analyses.
| |
Acknowledgments |
|---|
We would like to thank Bart E. Laurijssens for assistance with development of the equilibrium dialysis method.
| |
Footnotes |
|---|
Accepted for publication January 6, 2000.
Received for publication October 5, 1999.
1 This work was supported by Grants MH-34223, DA-05258, MH-19924, and RR-00054 from the Department of Health and Human Services. L.L.v.M. is the recipient of a Scientist Development Award (K21-MH-01237) from the National Institute of Mental Health, National Institutes of Health.
Send reprint requests to: David J. Greenblatt, M.D., Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. E-mail: Dj.Greenblatt{at}tufts.edu
| |
References |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  K. Venkatakrishnan and R. S. Obach IN VITRO-IN VIVO EXTRAPOLATION OF CYP2D6 INACTIVATION BY PAROXETINE: PREDICTION OF NONSTATIONARY PHARMACOKINETICS AND DRUG INTERACTION MAGNITUDE Drug Metab. Dispos., June 1, 2005; 33(6): 845 - 852. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. P. Austin, P. Barton, S. Mohmed, and R. J. Riley THE BINDING OF DRUGS TO HEPATOCYTES AND ITS RELATIONSHIP TO PHYSICOCHEMICAL PROPERTIES Drug Metab. Dispos., March 1, 2005; 33(3): 419 - 425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. Jones and J. B. Houston SUBSTRATE DEPLETION APPROACH FOR DETERMINING IN VITRO METABOLIC CLEARANCE: TIME DEPENDENCIES IN HEPATOCYTE AND MICROSOMAL INCUBATIONS Drug Metab. Dispos., September 1, 2004; 32(9): 973 - 982. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. H. Court, S. Krishnaswamy, Q. Hao, S. X. Duan, C. J. Patten, L. L. von Moltke, and D. J. Greenblatt EVALUATION OF 3'-AZIDO-3'-DEOXYTHYMIDINE, MORPHINE, AND CODEINE AS PROBE SUBSTRATES FOR UDP-GLUCURONOSYLTRANSFERASE 2B7 (UGT2B7) IN HUMAN LIVER MICROSOMES: SPECIFICITY AND INFLUENCE OF THE UGT2B7*2 POLYMORPHISM Drug Metab. Dispos., September 1, 2003; 31(9): 1125 - 1133. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Margolis and R. S. Obach Impact of Nonspecific Binding to Microsomes and Phospholipid on the Inhibition of Cytochrome P4502D6: Implications for Relating in Vitro Inhibition Data to in Vivo Drug Interactions Drug Metab. Dispos., May 1, 2003; 31(5): 606 - 611. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Lu, X. Meng, C. Li, S. Sang, C. Patten, S. Sheng, J. Hong, N. Bai, B. Winnik, C.-T. Ho, et al. Glucuronides of Tea Catechins: Enzymology of Biosynthesis and Biological Activities Drug Metab. Dispos., April 1, 2003; 31(4): 452 - 461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. H. Tran, L. L. von Moltke, K. Venkatakrishnan, B. W. Granda, M. A. Gibbs, R. S. Obach, J. S. Harmatz, and D. J. Greenblatt Microsomal Protein Concentration Modifies the Apparent Inhibitory Potency of CYP3A Inhibitors Drug Metab. Dispos., December 1, 2002; 30(12): 1441 - 1445. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. P. Austin, P. Barton, S. L. Cockroft, M. C. Wenlock, and R. J. Riley The Influence of Nonspecific Microsomal Binding on Apparent Intrinsic Clearance, and Its Prediction from Physicochemical Properties Drug Metab. Dispos., December 1, 2002; 30(12): 1497 - 1503. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-S. Wang, X. Wen, J. T. Backman, and P. J. Neuvonen Effect of Albumin and Cytosol on Enzyme Kinetics of Tolbutamide Hydroxylation and on Inhibition of CYP2C9 by Gemfibrozil in Human Liver Microsomes J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 43 - 49. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Tang, Y. Lin, A. D. Rodrigues, and J. H. Lin Effect of Albumin on Phenytoin and Tolbutamide Metabolism in Human Liver Microsomes: An Impact More Than Protein Binding Drug Metab. Dispos., June 1, 2002; 30(6): 648 - 654. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Hutzler and T. S. Tracy Atypical Kinetic Profiles in Drug Metabolism Reactions Drug Metab. Dispos., April 1, 2002; 30(4): 355 - 362. [Full Text] [PDF]
|
|
|
|
|
|
J. C. Kalvass, D. A. Tess, C. Giragossian, M. C. Linhares, and T. S. Maurer Influence of Microsomal Concentration on Apparent Intrinsic Clearance: Implications for Scaling in Vitro Data Drug Metab. Dispos., October 1, 2001; 29(10): 1332 - 1336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Hemeryck, C. A. De Vriendt, and F. M. Belpaire Metoprolol-Paroxetine Interaction in Human Liver Microsomes: Stereoselective Aspects and Prediction of the in Vivo Interaction Drug Metab. Dispos., April 13, 2001; 29(5): 656 - 663. [Abstract] [Full Text]
|
|
|
|
|
|
K. Venkatakrishnan, L. L. von Moltke, and D. J. Greenblatt Application of the Relative Activity Factor Approach in Scaling from Heterologously Expressed Cytochromes P450 to Human Liver Microsomes: Studies on Amitriptyline as a Model Substrate J. Pharmacol. Exp. Ther., April 1, 2001; 297(1): 326 - 337. [Abstract] [Full Text]
|
|
|
|
|
|
L. M. Hesse, K. Venkatakrishnan, L. L. von Moltke, R. I. Shader, and D. J. Greenblatt CYP3A4 Is the Major CYP Isoform Mediating the in Vitro Hydroxylation and Demethylation of Flunitrazepam Drug Metab. Dispos., February 1, 2001; 29(2): 133 - 140. [Abstract] [Full Text]
|
|
|
|
|
|
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]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
