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Vol. 280, Issue 3, 1392-1400, 1997
Analytical and Metabolic Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan (T.I., K.H., S.E., K.S.), and Faculty of Pharmaceutical Science, University of Tokyo, Tokyo, Japan (Y.S.)
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Abstract |
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 Top
Abstract
Introduction
Methods
Results
Discussion
References
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Sulfation and glucuronidation, the major routes of metabolism of troglitazone, an antidiabetic agent, were examined in vitro using hepatic cytosol and microsomes prepared from KK mice, an animal model of non-insulin-dependent diabetes mellitus. Stereoselectivity was observed for both conjugation reactions, and the metabolic intrinsic clearance of glucuronidation was about 3- to 100-fold higher than that of sulfation for each stereoisomer. In addition, the metabolic intrinsic clearance of glucuronidation exhibited an 8-fold difference among stereoisomers. The predicted metabolic clearance for each stereoisomer, calculated from the in vitro data based on a dispersion model, was comparable to the measured metabolic clearance in vivo, ranging from 27 to 93%. We also attempted to predict the in vivo metabolic clearance from in vitro metabolism data, to investigate species differences in the stereoselectivity of the conjugation reactions in normal animals, i.e., ddY mice and rats. For ddY mice the in vivo hepatic glucuronidation clearance was 170-fold higher than the corresponding sulfation clearance, whereas for rats the sulfation clearance was 6-fold higher than the glucuronidation clearance. The hepatic sulfation clearance in mice and rats predicted from in vitro metabolism data was 5.3- and 1.1-fold higher, respectively, than that in vivo, calculated from the plasma disposition of parent drug and biliary excretion of metabolites. For glucuronidation, the predicted values in mice and rats were 1.0- and 0.33-fold higher, respectively. These results suggest that semiquantitative extrapolation of in vitro stereoselective metabolism of troglitazone, by conjugation, to the in vivo situation is possible.
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Introduction |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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To extrapolate in
vitro metabolism data to the in vivo situation, the
following approach is increasingly used: estimation of enzyme kinetic
parameters per unit liver weight from in vitro metabolism
data, extrapolation of these data to the metabolizing organ and whole
body using an appropriate mathematical model based on clearance
concepts and physiological pharmacokinetics and comparison of the
prediction with the drug's pharmacokinetics observed in vivo. This approach has been successfully used for many drugs (Rane et al., 1977
; Roberts and Rowland, 1986a; Wilkinson,
1987; Sugiyama et al., 1989; Houston, 1994; Iwatsubo
et al., 1996).
Troglitazone (CS-045) is a novel oral antidiabetic agent with
hypoglycemic activity in non-insulin-dependent diabetes mellitus in
animals and humans (Fujiwara et al., 1988; Suter et
al., 1992). Troglitazone is an equal mixture of four
stereoisomers, arising from two asymmetric carbons at the 2-position of
the chroman ring and the 5-position of the thiazolidine ring (fig.
1). Little unchanged drug is excreted in the urine or
bile (Kawai et al., in press) and, therefore, the
CLtot of the drug is determined by its metabolic clearance (Izumi et al., 1996). Furthermore, because
troglitazone is mainly metabolized in liver,
CLtot essentially reflects hepatic metabolism
(Izumi et al., 1996). Glucuronidation and sulfation are the
major pathways of metabolism, along with a minor degree of oxidation to
the quinone form (Kawai et al., in press).
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We previously demonstrated stereoselective troglitazone pharmacokinetics in KK mice, an animal model of non-insulin-dependent diabetes mellitus, that involved epimerization at the 5-position of the thiazolidine ring and metabolic clearance (Izumi et al., in press). In the present study, the stereoselectivity in metabolic clearance involving the sulfate and glucuronide conjugation of troglitazone stereoisomers was investigated using hepatic cytosol and microsomal fractions, respectively. Subsequently, the in vitro kinetic parameters were used to predict the in vivo clearance of the drug, which was then compared with that determined experimentally in vivo. The same approach was also used to examine species differences in both conjugation reactions in normal ddY mice and rats.
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Methods |
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Abstract
Introduction
Methods
Results
Discussion
References
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Chemicals
Troglitazone and its four stereoisomers (SS,
SR, RS and RR) were synthesized by
Sankyo Co. (Tokyo, Japan) (Yoshioka et al., 1989). The
sulfate (Yoshioka et al., 1987) and glucuronide conjugates (Yoshioka et al., 1991) were also synthesized by Sankyo Co.
PAPS and UDPGA were purchased from Sigma Chemical Co. (St. Louis, MO) and Seikagaku Kogyo (Tokyo, Japan), respectively. The other reagents and solvents used were of analytical and HPLC grade, respectively.
Animals
Adult male KK mice (25-35 g) and ddY mice (25-35 g) were purchased from Tokyo Experimental Animals (Tokyo, Japan) and Japan SLC (Shizuoka, Japan), respectively. Adult male Wistar-Imamichi rats (300-350 g) were purchased from the Imamichi Institute for Animal Reproduction (Saitama, Japan). The animals were allowed free access to food and drink for more than 1 week; immediately before the experiments, each animal was fasted overnight.
Preparation of Hepatic Cytosol and Microsomes
Cytosolic (Sekura and Jakoby, 1979) and microsomal (Kutt and
Fouts, 1971) fractions were prepared according to standard procedures and stored at 
80°C until used. Protein concentration was determined by the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL),
using human serum albumin (standard solution; Sigma) as a standard.
Measurement of Conjugating Activities In Vitro
Cytosolic sulfation.
After 100 µl of mouse liver cytosol
(final concentration, 2 mg/ml) and 375 µl of Tris-HCl buffer (pH 7.2, 0.1 M) containing 5 mM 2-mercaptoethanol were kept at 37°C for 10 min, 25 µl of PAPS solution (final concentration, 200 µM) (Pacifici
et al., 1988) and 5 µl of troglitazone stereoisomer in
N,N
-dimethylformamide solution (final
concentration, 0.25-50 µM) were added to start the sulfation
reaction. The reaction was stopped after 10 min by mixing with an
ethanolic solution of 9-acetylanthracene (used as an analytical
internal standard). Preliminary studies showed that sulfation was
linear with respect to both time and protein concentration over this
incubation period. In the studies with rat hepatic cytosol, the protein
concentration was 0.5 mg/ml and the reaction time was 3 min; other
incubation conditions were the same as in the mouse studies.
Microsomal glucuronidation.
A suspension of mouse or rat
liver microsomes (10 mg/ml) was mixed with 50 mM Tris-HCl (pH 7.4)
buffer containing 10 mM MgCl2 and Brij 58 (0.2 mg/mg
microsomal protein) and was incubated for 30 min under ice-cooled
conditions to activate the microsomes (Lett et al., 1992).
The activated 100-µl microsomal sample (final concentration, 2 mg/ml)
was mixed with 375 µl of 0.1 M Tris-HCl buffer (pH 7.4) containing 5 mM MgCl2 and 2 mM D-saccharic acid 1,4-lactone
(El-Mouelhi and Bock, 1991) and was preincubated at 37°C for 10 min;
then 25 µl of UDPGA solution (final concentration, 5 mM) and 5 µl
of troglitazone solution in
N,N-dimethylformamide (final concentration,
0.25-50 µM) were added to initiate glucuronidation. Preliminary
experiments indicated that the formation of glucuronide was linear up
to 10 min; therefore, the reaction was terminated at 5 min by addition
of ethanol.
Determination of sulfate and glucuronide. An LC-10A HPLC system (Shimadzu, Kyoto, Japan) was used. The column for the analysis of sulfate conjugates was a YMC-Pack, ODS-A, A-314G column (YMC, Kyoto, Japan), and the mobile phase was acetonitrile/water/phosphoric acid (62:38:0.05, v/v); the detection wavelength was 230 nm, the flow rate was 1.2 ml/min and the column temperature was 35°C. The column for the analysis of glucuronides was a YMC-Pack, ODS-A, A-312 column (YMC). The mobile phase, detection wavelength, flow rate and column temperature were acetonitrile/water/phosphoric acid (45:55:0.05, v/v), 230 nm, 1.2 ml/min and 35°C, respectively. The limit of detection for sulfate and glucuronide conjugates was 60 nM.
Determination of fu in
cytosolic and microsomal samples.
The fu of
troglitazone stereoisomers in cytosolic and microsomal incubations at
37°C was determined using a HPFA method (Shibukawa et al.,
1995). Preliminary experiments indicated that the measurement of the
fu of stereoisomers in the cytosol required a
large volume to be injected onto the HPFA column and the plateau phase
was short because of the high Cu. Under linear
binding conditions, the reciprocal of fu should
be proportional to the protein concentration. Therefore, for cytosolic
binding, the 1/fu value at 2 mg/ml cytosol (0.5 mg/ml for rat cytosol) after addition of 2S-isomers (equal mixture of SS and SR) and 2R-isomers
(equal mixture of RS and RR) at 2 and 50 µM was
extrapolated by linear regression from those at 10, 20 and 40 mg/ml
cytosol, without the addition of PAPS. For microsomes, 2 or 50 µM
troglitazone 2S- or 2R-isomers was added to 2 mg/ml microsomal suspension; the other incubation conditions were the
same except for the absence of UDPGA. The total concentration in the
microsomal mixture was measured by HPLC as described above. After
centrifugation at 105,000 × g for 30 min at 4°C, the
supernatant concentration was measured by HPLC (Izumi et
al., 1996). For measurement of Cu, 3 ml of
cytosolic or microsomal supernatant was injected onto the HPFA column
(Develosil 100Diol5; Nomura Chemical Co., Aichi, Japan), which was
equilibrated with phosphate buffer (pH 7.4, ionic strength of 0.17) at
a flow rate of 1 ml/min at 37°C. The plateau region of the
troglitazone peak (i.e., troglitazone
fu) was delivered to the preconcentration column
(Develosil ODS10; Nomura Chemical Co.) for 5 min on each occasion,
using a column-switching apparatus (FCV-12AH; Shimadzu). The unbound
drug eluted with the mobile phase (acetonitorile/water/phosphoric acid,
55:45:0.1, v/v) was analyzed on an analytical column (Cosmosil 5C18-AR;
Nacalai Tesque, Kyoto, Japan).
Recovery from cytosol and microsomes.
The conversion values
to calculate the kinetic parameters per unit liver weight were
evaluated from the total cytosolic protein per unit liver weight,
calculated from the total volume and protein concentration in cytosol,
assuming 100% recovery. The cytosolic protein recoveries for KK mice,
ddY mice and rats were 65.8, 52.3 and 50.7 mg/g liver, respectively.
The microsomal protein recovery (Lin et al., 1980) was
determined from the total content of cytochrome P450, a marker enzyme
for microsomes, in the homogenate (Matsubara et al., 1976)
and microsomes (Omura and Sato, 1964). The values for KK mice, ddY mice
and rats were 53.2, 40.8 and 45.4 mg/g liver, respectively.
Calculation of Enzyme Kinetic Parameters and CLuint
The enzyme kinetic parameters for the sulfation and glucuronidation of the troglitazone stereoisomers were estimated using the Cu value, measured by the HPFA method. Depending on whether Eadie-Hofstee plots show curvature or not, the data were analyzed by equation 1 (linear plot) or equation 2 (curvature).
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(1) |
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(2) |
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(3) |
), using
the v and Cu values with a weighting of 1/(Cu)2. Under linear conditions,
CLuint could be calculated from equation 4.
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(4) |
), the
CLH can be calculated from equations 5 and 6.
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(5) |
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(6) |
) and a value of 0.17 was used (Roberts and Rowland,
1986a; Sugiyama et al., 1989; Iwatsubo et al.,
1996). CLH was calculated from equations 5 and 6
by substituting the sum of CLuint,Sulf. and
CLuint,Glu. for each stereoisomer. CLH,Sulf. and CLH,Glu.
were further calculated from equations 7 and 8.
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(7) |
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(8) |
Biliary and Urinary Excretion of Troglitazone in ddY Mice and Rats
Under urethane anesthesia, ddY mouse gallbladder was ligated, and the bile duct was cannulated using PE-10 tubing. After the bile flow rate was confirmed to be constant, troglitazone (5 mg/kg) was administered via the jugular vein, and bile was collected for 6 hr after administration. Excreted urine was collected, as well as urine that remained in the bladder, for 6 hr after administration. Blood was collected from the carotid artery of separate uncannulated mice at the times stated.
In rats, the bile duct was cannulated, under urethane anesthesia, using PE-50 tubing. Blood was collected from a similar cannula placed in the femoral artery. Under urethane anesthesia, the blood and bile were collected at the times stated for 6 hr after administration of troglitazone (5 mg/kg) via the jugular vein. Excreted urine and that remaining in the urinary bladder were collected.
Determination of Troglitazone and Its Sulfate and Glucuronide Concentrations
Troglitazone and the sulfate conjugate in plasma were extracted with ethyl acetate/n-hexane (90:10, v/v) after addition of tetrabutylammonium phosphate (Waters, Milford, MA) and internal standard solution (9-acetylanthracene) and were analyzed by HPLC. Troglitazone glucuronide in plasma was extracted with a Bond Elut cartridge (CBA; Analytichem International, Harbor City, CA); after washing with acetate buffer (pH 6.0, 0.1 M) and chloroform, and adding internal standard (9-anthracene carboxylic acid), and the glucuronide was eluted with ethanol. Troglitazone and sulfate conjugate in bile and urine were extracted with ethanol containing internal standard (9-acetylanthracene) and were analyzed by HPLC. Glucuronide conjugate in bile or urine was extracted using a Bond Elut cartridge (CBA; Analytichem International); after washing with acetate buffer containing 0.1% Triton X-100, acetate buffer and chloroform and adding internal standard (9-anthracene carboxylic acid), and the glucuronide was eluted with ethanol and analyzed by HPLC. The HPLC conditions were the same as described above.
Estimation of the Pharmacokinetic Parameters of Troglitazone in ddY Mice and Rats
The plasma profiles of troglitazone after i.v. administration to
ddY mice and rats were fitted to a biexponential equation (eq. 9),
using a nonlinear least-squares method (MULTI), to obtain A,
B, 
and 
with a weighting of
1/(concentration)2. The CLtot was
calculated from equation 10. Because the CLtot of troglitazone is assumed to primarily reflect hepatic metabolic clearance, the CLH for each conjugate was
calculated from the product of CLtot and the
fraction of each metabolite excreted in the urine and bile.
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(9) |
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(10) |
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(11) |
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Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Sulfation and glucuronidation in KK mouse liver cytosol and microsomes. The cytosolic binding, the 1/fu values, at each protein concentration in the cytosol showed good linearity (r > 0.92), and the extrapolated fu value at 2 mg protein/ml cytosol was 0.03 with 2 and 50 µM added 2S- and 2R-isomer concentrations (table 1). In microsomes, >70% of the stereoisomers was found to be adsorbed to the microsomes (2 mg protein/ml). The fu value in the 105,000 × g supernatant was 0.03. This binding in the supernatant was considered to mainly reflect that to Brij 58, which was used for activation of UDP-glucuronosyltransferase, because the fu value was similar to that at the same concentration of Brij 58 solution alone. In contrast, the cytosolic binding appeared to be due to binding to the cytosolic protein. From these results, the fu value for the stereoisomers in the reaction mixtures was estimated to be 0.01 at 2 and 50 µM added drug concentration (table 1).
|
) (fig.
2). For glucuronidation, the Eadie-Hofstee plots for the
four stereoisomers were monophasic and the enzyme kinetic parameters
were analyzed by equation 1 (fig. 2). The unbound plasma concentrations
of troglitazone stereoisomers in KK mice after i.v. administration of 5 mg/kg 2S- or 2R-isomers were estimated to be
<0.06 µM and were obtained by multiplying the total concentration by
a plasma fu value of 0.003 in KK mouse plasma
(Izumi et al., in press). Therefore, both conjugating
reactions would be expected to occur in a linear fashion over the range
of Cu studied. The CLuint,Sulf. for the S-configuration
at the 2-position in the chroman ring was about 4-fold greater than
that for the R-configuration (table 1). On the other hand,
the CLuint,Glu. for the
R-configuration at the 2-position of the chroman ring was
about 8-fold greater than that for the S-configuration
(table 1). The CLuint,Glu. per unit liver weight
(milliliters per minute per gram of liver) for each stereoisomer was
also higher than the CLuint,Sulf.,
i.e., 6-fold for SS, 3-fold for SR,
50-fold for RS and 100-fold for RR (table 1).
These results suggest that glucuronide conjugation is the major
metabolic pathway in KK mice.
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In vivo conjugative metabolic clearance in ddY mice and
rats.
The ratio of total-body blood clearance, i.e.,
CLtot divided by RB, to hepatic
blood flow (90 ml/min/kg for mice and 55 ml/min/kg for rats) (Davies
and Morris, 1993), reflecting the hepatic extraction ratio, was 0.36 for mice and 0.95 for rats, respectively (table 2). There was also a
species difference with respect to the metabolites present in plasma
(fig. 3). The plasma glucuronide levels were higher than
those of the sulfate in ddY mice; in contrast, for rats, the sulfate
was a major metabolite (fig. 3). No troglitazone or either conjugate
was detected in the urine of both species. Troglitazone was not found
in bile, and the amount of glucuronide conjugate excreted into bile was
63.5 ± 3.7% (mean ± S.D. of four experiments) of the
administered dose for ddY mice and 11.6 ± 0.6% for rats. The
corresponding figure for the sulfate conjugate excreted in bile was
0.373 ± 0.128% for ddY mice and 68.0 ± 0.8% for rats.
Therefore, for rats the amount of glucuronide excreted into bile was
170-fold greater than that of the sulfate, whereas for rats sulfate
excretion was about 6-fold higher than glucuronide excretion.
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In vitro stereoselective sulfation and glucuronidation
in ddY mice and rats.
For both species the
fu value in the cytosol and microsomes was small
and there were no large differences between the 2S- and
2R-isomers over the range of concentrations used (tables
3 and 4). The relationship between
v and Cu in ddY mice was very similar
to that for KK mice (fig. 4). For rats, substrate
inhibition of the formation of the sulfate conjugate was observed (fig.
5) and the Ki values
for 2S-isomers (0.054 and 0.070 µM) were lower than those
for the 2R-isomers (1.1 and 3.9 µM) (table 4). However, substrate inhibition was considered not to occur in vivo,
because the estimated maximum plasma Cu (0.02 µM) was less than the Ki values.
The maximum plasma Cu was estimated by
multiplying the total plasma concentration (fig. 3) by the plasma
fu value of 0.00092 in rat plasma (Izumi
et al., 1996).
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Stereoselectivity in the in vitro metabolism
experiments (KK mice).
The stereoselectivity in the sulfation and
glucuronidation of troglitazone were investigated using KK mouse liver
cytosol and microsomes. For in vitro-in vivo
scaling, the correction for the fu in the
reaction mixture has been reported to be important (Lin et
al., 1980; Houston, 1994; Obach, 1996). For troglitazone, high
protein binding in the reaction mixture was anticipated because of the
highly lipophilic nature of the drug. Therefore, the
fu in the reaction mixture of cytosol and
microsomes was measured by the HPFA method. The
fu of troglitazone stereoisomers in cytosol was
0.03 and that in the microsomal suspension was about 0.01, and these
values seemed to be constant over the range of concentrations studied
(table 1).
; Hansen et al., 1992; Pesola and Walle, 1993;
Walle et al., 1993). However, there have been no reports for
drugs, such as troglitazone, where stereoselective sulfation and
glucuronidation occur at the same phenolic hydroxyl group.
The sum of CLH calculated based on a dispersion
model using CLuint,Sulf. and
CLuint,Glu. for each stereoisomer, as obtained from in vitro metabolism experiments, was found to be
comparable to the CLH in vivo for
each stereoisomer (Izumi et al., in press), except for
SR (table 2). These results indicate that the in
vitro metabolism experiments reflect the in vivo
metabolism and that stereoselectivity in vivo is mainly due
to glucuronide conjugation. There have been a few reports of
extrapolation from in vitro experiments to the in
vivo situation for glucuronidated drugs; moreover, this situation
may be difficult because of the uncertain nature of the
rate-controlling factors, such as the concentration of co-substrate and
the condition of the enzymes (Rane et al., 1984; Mistry and Houston, 1987). In this study, the reaction conditions were set to
obtain maximum glucuronidation activity by the addition of co-substrate
(UDPGA), activation by detergent (Brij 58) and Mg++ and
inhibition of -glucuronidase by the addition of a saccharic acid
1,4-lactone. In particular, the UDP-glucuronosyltransferase in prepared
microsomes is known to be in a "latent" state (Burchell and
Coughtrie, 1989). The concentration of Brij 58 for activation of
UDP-glucuronosyltransferase was that reported for the glucuronidation of 1-naphthol and morphine (Lett et al., 1992).
Species differences in conjugative metabolism between normal mice and rats. We also examined the in vitro-in vivo extrapolation using normal mice, where glucuronidation is the major metabolic pathway, and rats, where sulfation is the major pathway. Substrate inhibition of sulfation was observed in rat liver cytosol. Such inhibition was not observed in sulfation by KK and ddY mouse liver cytosol, and the Km values in rats were very low, i.e., 1/10 to 1/130 of that in mice (tables 1, 3 and 4). These results demonstrate that sulfotransferases with different affinities for substrates are involved in the sulfation of troglitazone in mice and rats.
A higher CLuint, Sulf. was observed for the S-configuration of the 2-position of the chroman ring in mice, whereas a higher CLuint, Glu. was observed for the R-configuration (table 3). For rats, a higher CLuint of both conjugations was noted for the R-configuration (table 4). Thus, preferential sulfation of the R-configuration was observed in both species. Because a mixture of the four stereoisomers was used in the in vivo experiments, the in vitro hepatic metabolic clearance of both conjugative reactions was calculated, based on a dispersion model using the mean CLuint obtained from in vitro conjugation reactions for each stereoisomer and compared with each other (table 2). The differences in sulfation and glucuronidation between mice and rats observed in vivo could be semiquatitatively predicted by extrapolation from the in vitro metabolism experiments. Although the major pathways, CLH,Glu. for ddY mice and CLH,Sulf. for rats, were well predicted in the in vivo CLH, the minor pathway CLH,Sulf. for ddY mice was overpredicted and the minor pathway CLH,Glu. for rats was underpredicted (table 2). In this study, we used the dispersion model to predict CLH from the in vitro metabolism experiments based on our previous studies (Sugiyama et al., 1989; Iwatsbo et al., 1996), which indicated that the
dispersion model (with DN of 0.17) was more
appropriate than other models, such as the well-stirred model, for the
prediction of a highly oxidatively metabolized drug. In the present
study, the predicted CLH for RR and
RS in KK mice, CLH,Glu. for ddY mice and CLH,Sulf. for rats, which showed relatively
high clearance, were about 1.2-fold higher than those based on the
well-stirred model, and the values predicted from the dispersion model
were close to the CLH in vivo. On the
other hand, the predicted CLH for SS
and SR in KK mice, CLH,Sulf. for ddY
mice and CLH,Glu. for rats, which showed low
clearance, were almost identical for the two models. These results
suggest that the dispersion model may be appropriate also for the
prediction of in vivo conjugative CLH
from in vitro metabolism data.
Uneven distribution of sulfotransferase and UDP-glucuronosyltransferase
in the hepatic lobes in rats has been reported, with a periportal
localization of sulfation and uniform or perivenously distributed
glucuronidation (Pang et al., 1992). These uneven zonations
were estimated to affect competitive pathways of enzymes (Morris and
Pang, 1987). For troglitazone, sulfation and glucuronidation are
competitive pathways in KK and ddY mice and rats. However, no
correction for this uneven distribution of enzymes was carried out in
the present studies, because of a lack of quantitative information on
the localization of isozymes of conjugating enzymes for troglitazone in
liver. In rats, because the clearance is high and the enzyme reaction
is assumed to be linear, the actual CLH,Sulf. assuming zonation might, in fact, be larger than that estimated in the
present study.
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Footnotes |
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Accepted for publication November 22, 1996.
Received for publication February 6, 1996.
Send reprint requests to: Takashi Izumi, Analytical and Metabolic Research Laboratories, Sankyo Co., 2-58, Hiromachi 1-Chome, Shinagawa-ku, Tokyo 140, Japan.
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Abbreviations |
|---|
CLH, hepatic plasma
clearance;
CLH,Glu., hepatic plasma
clearance for glucuronidation;
CLH,Sulf., hepatic plasma clearance for sulfation;
CLtot, total-body plasma clearance;
CLuint, metabolic intrinsic clearance;
CLuint,Glu., metabolic intrinsic clearance
for glucuronidation;
CLuint,Sulf., metabolic
intrinsic clearance for sulfation;
Cu, unbound concentration;
fu, unbound fraction;
HPFA, high-performance frontal analysis;
HPLC, high-performance liquid
chromatography;
PAPS, adenosine-3-phosphate-5-phosphosulfate;
UDPGA, uridine-5-diphosphoglucuronic acid.
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References |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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T. B. Andersson, H. Sjöberg, K.-J. Hoffmann, A. R. Boobis, P. Watts, R. J. Edwards, B. G. Lake, R. J. Price, A. B. Renwick, M. J. Gómez-Lechón, et al. An Assessment of Human Liver-Derived in Vitro Systems to Predict the in Vivo Metabolism and Clearance of Almokalant Drug Metab. Dispos., April 13, 2001; 29(5): 712 - 720. [Abstract] [Full Text]
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H. Yamazaki, A. Shibata, M. Suzuki, M. Nakajima, N. Shimada, F. P. Guengerich, and T. Yokoi Oxidation of Troglitazone to a Quinone-Type Metabolite Catalyzed by Cytochrome P-450 2C8 and P-450 3A4 in Human Liver Microsomes Drug Metab. Dispos., November 1, 1999; 27(11): 1260 - 1266. [Abstract] [Full Text]
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, SS; 
,
SR; 
, RS; 
, RR.
Fitted curves were calculated from equation 
