In vitro glucuronidation of D-23129, a new anticonvulsant, by human liver microsomes and liver slices

Article abstract of DOI:10.1080/004982597240424

1. The metabolic profile of D-23129, a new anticonvulsant agent, was studied in vitro using human liver microsomes and fresh liver slices. 2. Oxidative metabolism appeared to be minimal with D-23129. The percent mean total radioactivity not associated with the parent compound recovered from oxidative metabolism studies from three individual liver donors was 0.7% ± 0.6 SD and was not significantly different from [¹⁴C]-D-23129 incubated with heat inactivated microsomes, mean = 0.5% ± 0.4 SD. 3. Phase II conjugation dominated the metabolism of D-23129 producing two distinct N-glucuronides as the primary metabolites. These metabolites were identified by electrospray ionization LC/MS. 4. The apparent K(m), for one of the glucuronide metabolites was determined in human liver microsome preparations from two individual liver donors to be 131 and 264 μM respectively. V(max) determined for the same microsomal preparations yielded 48.9 and 59.9 pmol/min/mg protein.

Full text of DOI:10.1080/004982597240424

xenobiotica , 1997, vol. 27, no. 5, 431±441
glucuronidation of D-23129, a new
In vitro
anticonvulsant, by human liver microsom es and
liver slices
P. J. McNEILLY*‹, C. D. TORCHIN‹, L. W. ANDERSON‹,
I. M. KAPETANOVIC‹, H. J. KUPFERBERG‹
and J. M. STRONG‹
‹
Laboratory of Clinical Pharmacology, Oæce of Pharmaceutical Sciences, Center for
Drug Evaluation and Research, US Food and Drug Administration, Laurel, MD 20708,
USA
‹
Preclinical Pharmacology Section, Epilepsy Branch, National Institute of Neurological
Disorders and Stroke, National Institutes of Health, Rockville, MD 20857, USA
Received 4 November 1996
1. The metabolic pro®le of D-23129, a new anticonvulsant agent, was studied in vitro
using human liver microsomes and fresh liver slices.
2. Oxidative metabolism appeared to be minimal with D-23129. The percent mean
total radioactivity not associated with the parent compound recovered from oxidative
±
±
metabolism studies from three individual liver donors was 0 7 %³0 6 SD and was not
"%
signi®cantly diåerent from [ C]-D-23129 incubated with heat inactivated microsomes,
±
±
mean ¯ 0 5 %³0 4 SD.
3. Phase II conjugation dominated the metabolism of D-23129 producing two distinct
N-glucuronides as the primary metabolites. These metabolites were identi®ed by
electrospray ionization LC MS.
}
4. The apparent K for one of the glucuronide metabolites was determined in human
lm
m
liver microsome preparations from two individual liver donors to be 131 and 264
±
determined for the same microsomal preparations yielded 48 9 and
respectively. V
max
59 9 pmol min mg protein.
±
}
}
Introduction
Epilepsy comprises many diverse disorders and syndromes with heterogeneous
aetiologies and neuropathologies. It is a chronic disease and is estimated to aåect
"
1±2 % of the worlds population ( 50 million persons), which makes it one of the
most common neurological disorders (Porter 1993). A large percentage (20±30%) of
patients diagnosed as having epilepsy are not adequately controlled by presently
available medications. The therapy generally involves a life-long polypharmacy and
drug±drug interactions are not uncommon (Rall and Schleifer 1990).
D-23129 [N-(2-amino-4-(4-¯uorobenzylamino)-phenyl)carbamic acid ethyl
ester] is a new and promising experimental anticonvulsant compound which is
eåective in most in vitro and in vivo models of epilepsy and is reported to have
multiple mechanisms of action (®gure 1) (Kapetanovic et al. 1995, Yonekawa
et al. 1995, Kapetanovic and Rundfeldt 1996, Rostock et al. 1996). It
was synthesized in 1988 by ASTA Medica AG (Frankfurt, Germany) as a desazo
analogue of a centrally acting, non-opioid analgesic drug, ¯upirtine. D-23129
evolved out of quantitative structure±activity studies and molecular modelling
initiated at ASTA Medica AG to separate the analgesic and anticonvulsant activity
(Seydel et al. 1994). It was selected for further development as an anticonvulsant
* Author for correspondence.
±
’
0049±8254}97 $12 00
1997 Taylor & Francis Ltd

432
P. J. McNeilly et al.
Figure 1. Structure of D-23129 and Flupirtine.
drug due to its greater potency and more favourable therapeutic index than
¯upirtine. D-23129 contains a phenyl in lieu of pyridinyl moiety of ¯upirtine.
It is important to characterize the metabolic pro®le of new candidate therapeutic
agents early during the drug development and prior to the administration to man. In
the past, various animal species have been used to establish drug metabolic pro®les.
However, the use of human liver tissue for drug metabolism studies in vitro avoids
interspecies diåerences in metabolism and has the potential to predict possible
drug±drug interactions and toxicity during the preclinical drug development phase.
It has been reported that the analog of D-23129, ¯upirtine, is extensively
metabolized (Obermeier et al. 1985, Hlavica and Niebch 1985), therefore the aim of
the present study was to investigate the phase 1 and 2 metabolism of this promising
new anticonvulsant drug, D-23129, using both human liver microsomes and liver
slices in vitro.
M aterials and m ethods
Chemicals and reagents
"
D-23129 ( 99 7 %), [aniline ring-U-"% C]-D-23129 (121 mCi mmol), AWD21-360 [N-(2-amino-4-
(4-¯uorobenzylamino)acetamide], AWD21-361 [1,2-diacetylamino-4-(4 -¯uorobenzylamino)benzol],
±
}
«
D-22939 [5-(4-¯uorobenzylamino)-1,3-dihydrobenzimidazole-2-one], ZP3 [1,2-diamino-4-(4-¯uoro-
benzylamino)benzol] and p-¯uorohippuric acid were obtained from Arzneimittelwerk Dresden GmbH
±
(Radebeul, Germany). The radioactive material was 97 6% pure as determined by hplc analysis.
+
«
Uridine-5 -diphosphoglucuronic acid (UDPGA), bovine serum albumin (BSA), NADP , glucose 6-
phosphate, glucose 6-phosphate dehydrogenase, dimethylsulphoxide, magnesium chloride hexahydrate,
b d
- -glucuronidase and Krebs±
saccharolactone (glucosaccharo 1,4-lactone), 2-mercaptoethanol,
Henseleit buåer were obtained from Sigma Chemical Co. (St Louis, MO, USA). Methyl-t-butyl ether
(MTBE) was obtained from J. T. Baker (Phillipsburg, NJ, USA). All other solvents were of hplc of
analytical grade.
Human liver specimens, medically unsuitable for transplantation, were acquired under the auspices
of the Washington Regional Transplant Consortium (Washington, DC, USA). These represented ®ve
specimens from three males and two females who ranged in age from 35 to 73 years. The two female
donors were smokers and four of the ®ve donors had anti-CMV positive serologies. All the donors were
reported to be social drinkers and one male was known to be a cocaine abuser. One male donor chronically
took diltiazem, another male was chronically taking captopril and aspirin and one female was reported to
±
l
be taking diazepam. Fresh human liver slices (300 m¬0 1 cm) were acquired through In Vitro
Technologies (Baltimore, MD, USA) (Harris et al. 1994) from liver samples obtained by the International
Institute for the Advancement of Medicine (Exton, PA, USA).

In vitro glucuronidation
433
Human liver microsome preparation
°
Human liver samples were obtained and stored at ®80 C until use. Microsomes were prepared by
tissue homogenization and diåerential centrifugation as previously described (Jamis-Dow et al. 1995).
Protein concentration was determined by the Bio-Rad method (Hercules, CA, USA) and microsomes
°
were aliquoted into vials and stored at ®80 C prior to use.
Microsomal and liver slice incubations
Oxidative metabolism was investigated by incubating
"%
lm
)
microsomal protein in (1 ml total volume) phosphate buåer containing 31 m KH PO , 69 m
[
C]-D-23129 (10±250
with 1 mg
m
m
#
%
±
m
m
l
Na HPO , 5 m MgCl and 1 m EDTA at pH 7 4. The reaction was started by addition of 10 l
#
%
#
+
m
m
NADPH generating system (10 m glucose 6-phosphate, 1 m NADP and 1 Sigma unit glucose 6-
phosphate dehydrogenase). Incubations were allowed to proceed for up to 1 h at which time the mixture
was placed on ice and extracted with 2 ml MTBE. A 1-ml aliquot of organic phase was removed and taken
l
l
to dryness under nitrogen, then reconstituted with 100 l hplc mobile phase. A 50- l portion of this
sample was injected onto hplc for analysis.
Microsomal glucuronidation was studied by incubating [ C]-D-23129 dissolved in dimethyl-
"%
±
sulphoxide with 1 mg microsomal protein in 250 m Tris-HCl buåer, 40 m MgCl , 2 8 m 2-
m
m
m
#
mercaptoethanol and 2 9 % (w v) BSA at pH 8 0. Final substrate concentrations were 50±2000
±
total volume of 1 ml. The reaction was started by the addition of 2 5 m UDPGA and allowed to proceed
±
lm
in a
}
±
m
°
for 120 min at 37 C. Incubations were halted by addition of 1 ml acetonitrile followed by centrifugation
°
l
(2100 g) at 4 C for 10 min. Portions (50 l) of the supernatant were analysed by hplc for presence of D-
23129 metabolites. Control experiments were performed in the absence of UDPGA or with microsomes
which had been heated in a boiling water bath for 5 min.
±
±
l
Fresh human liver slices (300 m¬0 1 cm) were allowed to preincubate in Krebs±Henseleit-2 25 %
(w v) BSA buåer for 1 h, at which point slices were transferred to wells (two slices per well) containing
}
±
fresh buåer in a total volume of 1 0 ml. D-23129 concentrations were 10 and 100
10 Ci ml ["% C] respectively. Reactions were started by addition of D-23129 and allowed to proceed for
lm
using 1 and
l
}
±
up to 4 h. Alternatively, 10 liver slices were incubated in 5 ml Krebs±Henseleit-2 25 % (w v) BSA
buåer at varying ["% C]-D-23129 concentration (10±100
carried out using buåer without liver slice and slices in the absence of substrate.
}
lm
l
, 1±10 Ci ml). Control experiments were
}
Hplc analysis
The chromatographic system consisted of a Hewlett Packard Series 1050 (Palo Alto, CA, USA) hplc
equipped with a quaternary pump, autosampler, diode array detector and a Packard Series A-100
Radiomatic Flo-One Beta detector (Meriden, CT, USA). Separation of D-23129 and its metabolites was
}
±
l
performed using a Vydac C4 reverse phase column (4 6¬250 mm, 5 m particle size) (Hesperia, CA,
±
USA) with a mobile phase of 50 m ammonium phosphate pH 5 0, 10 m NaCl, 100 EDTA
m
m
lm
}
±
acetonitrile 78 :22 (v v). The ¯ow rate was 1 0 ml min and D-23129 and its metabolites were monitored
at 220 nm. The analysis of ["% C] samples utilized a 1 0 ml ¯ow cell and used Flo Scint IV (Packard
}
}
±
Meriden, CT, USA) as scintillant. The scintillant to mobile phase ratio was 3 :1. Modi®cation of the hplc
±
mobile phase to 50 m ammonium formate pH 5 0 acetonitrile 78 : 22 (v v) was required to facilitate
m
}
}
identi®cation of the D-23129 conjugates by liquid chromatography electrospray ionization mass
spectrometry (LC ESI MS). The latter hplc conditions were used for analysis of the microsomal
}
}
}
}
glucuronide formation experiments and human liver slice incubations.
LC MS analysis
}
Mass spectral analysis was performed using a Finnigan TSQ 7000 equipped with an electrospray
interface (Finnigan MAT, San Jose, CA, USA). Separations were performed using a Hewlett Packard
1050 hplc system equipped with an autosampler and variable wavelength detector. MS MS experiments
}
±
were performed using argon 0 8 millitorr as collision induced dissociation (CID) gas with an applied
oåset voltage of 20±50 V.
Results
"%
Phase I metabolism of [ C]-D-23129 by human liver microsomes
Microsomal preparations from three individual human liver donors were
"%
incubated with [ C]-D-23129 and the media from these experiments were analysed
by hplc to evaluate the oxidative metabolism of this compound. Table 1 lists the total
radioactivity not associated with the parent compound and reported as a percent of
"%
the total [ C] injected onto the hplc column. The percent radioactivity recovered

434
P. J. McNeilly et al.
Table 1. Non-D-23129 radioactivity detected by hplc.
% Total injected radioactivity
lm
)
Control"
SD$
D-23129 (50
Avg SD
Liver
Avg#
%CV%
%CV
±
±
±
±
±
±
22 88
±
21 88
±
10 07
HL8
HL6
HL10
0 29
0 08
22 20
0 41
0 12
±
±
±
±
±
1 10
0 01
1 09
1 50
0 30
±
0 11
±
0 03
±
23 36
±
0 23
±
0 02
lm
" 50
#
D-23129 (heat-inactivated microsomes).
Mean of three determinations.
$ SD, standard deviation.
%
%CV, percent coeæcient of variation.
Figure 2. Hplc separation of D-23129 metabolites from human liver microsomes in the presence of
lm
UDPGA. Radiochromatogram of D-23129 (50
) after incubation with human liver micro-
somes: (A) aqueous fraction after MTBE extraction, (B) MTBE extract, and (C) MTBE extract
of heat inactivated microsome control.
from the hplc that was not associated with the parent compound was similar when
"%
[ C]-D-23129 was incubated with either active microsomes (mean ¯
±
±
±
±
0 7 %³0 6 SD) or heat-inactivated microsomes (mean ¯ 0 5 %³0 4 SD). When
"%
[ C]-D-23129 concentrations ranging from 10 to 250 lm and incubation times of
±
0 5±2 h were analysed, no radiolabelled peaks associated with enzymatic metabolism
of the parent compound were apparent (data not shown).
Phase II conjugation of D-23219 by human liver microsomes and slices
"%
Glucuronidation reactions were investigated by incubating [ C]-D-23129
(50 lm) with human microsomal preparations from three individual liver donors in
the presence of UDPGA followed by hplc analysis of the media. A typical hplc
radiochromatogram from these experiments is shown in ®gure 2 for (A) the aqueous
phase after extraction of the medium with MTBE, (B) the MTBE extract, and (C)

In vitro glucuronidation
435
lm
Figure 3. Incubation of ["% C] D-23129 (50
radiochromatograms of incubation media with (A) liver slice and (B) control without liver slice.
)
with human liver slices for 2 h showing hplc
a MTBE organic extract from a incubation containing heat inactivated microsomes.
"%
Figure 2A identi®ed two [ C]-D-23129 derived radioactive peaks with hplc
±
retention times of 5 1 and 7 6 min, more polar than the parent compound. These
±
were not observed in the MTBE extracts shown in ®gure 2B and C. The latter two
"%
tracings revealed a number of minor [ C]-D23129 derived radioactive peaks similar
to those observed for incubations containing either active or heat inactivated
microsomal preparations in the absence of UDPGA (see data in table 1). The peaks
±
±
with retention times of 5 1 and 7 6 min were observed in all three liver microsomal
preparations and accounted for 84 % of the non-parent drug related radioactivity
±
recovered from the hplc analysis. The peak at 29 0 min (®gure 2A) most likely
"%
represents unextracted [ C]-D-23129 or hydrolysis of the [ C]-D-23129 conjugate
"%
to the parent compound.
"%
Metabolism of [ C]-D-23129 was investigated in human liver slices from four
human liver donors to establish whether glycine or acetylated metabolites were
formed, similar to that observed for ¯upirtine (Obermeier et al. 1985, Hlavica and
Niebch 1985). A modi®ed hplc mobile phase as described in the Materials and
methods was used for the analysis of these experiments. Typical hplc radio-
"%
chromatograms of media from 2-h incubations of [ C]-D-23129 (50 lm) with or
without human liver slices are shown in ®gure 3A and B respectively. A major
±
radioactive peak with a hplc retention time of 11 3 min was noted as well as
radioactivity detected at retention times ranging from 5 to 7 min (®gure 3A). These
radioactive peaks were not present in control incubations (®gure 3B). The hplc
retention time of the synthesized potential metabolites, AWD21-360, AWD21-361,
ZP3 and p-¯uorohippuric acid, similar to that observed for ¯upirtine metabolism
did not correspond to any of the radioactive peaks observed in ®gure 3A (data not
shown).

436
P. J. McNeilly et al.
b
Figure 4. Eåect of -glucuronidase on the metabolites of D-23129. Hplc chromatograms showing UV
(220 nm) tracings of (A) D-23129 microsomal incubation medium prior to -glucuronidase
b
±
treatment, (B) in the presence of -glucuronidase and saccharolactone (12 5 m ), and (C) in
presence of -glucuronidase only. -Glucuronidase incubations were carried out in 100 m
b
m
b
b
m
±
phosphate buåer at pH 6 8 for 1 h.
Identi®cation of D-23129 N-glucuronides
Biochemical evidence for D-23129 glucuronides were obtained after incubation
of D-23129 with human liver microsomes in the presence of UDPGA. Hplc
chromatograms of the media obtained from these experiments using UV detection
±
are shown in ®gure 4. A major peak with a retention time of 11 3 min was observed
in ®gure 4A, however, the earlier eluting peak observed in the radiochromatograms,
®gures 2A and 3A, was obscured in ®gure 4A by UV absorbing interferences. The
±
peak eluting at 11 3 min (®gure 4A) decreased when the medium was treated with b-
glucuronidase (®gure 4C) and this reaction was inhibited by the addition of
saccharolactone to the b-glucuronidase incubation (®gure 4B). This provides
evidence for the formation of a D-23129 glucuronide.
Hplc chromatogram fractions ranging from 5 to 6 min and the peak eluting at
±
11 3 min were collected and analysed by LC ESI MS. Both positive and negative
}
}
ion electrospray spectra were obtained for these two hplc collections. Molecular ions
+
were observed in the positive ion (M-H) spectra at m z 480 and negative ion
}
Õ
(M-H) spectra at m z 478 respectively and were consistent with D-23129 glucu-
}
ronide conjugates. Positive ion product scans of the molecular ion for the two
conjugates shown in ®gure 5A and B were used to con®rm the identi®cation of the
two conjugates. With the exception of the ions at m z 208 (®gure 5A) and m z 206
}
}
(®gure 5B), both product scans were qualitatively similar. The ion observed at m z
}
371 was produced by loss of the p-¯uorobenzyl moiety from the molecular ion.
Electrospray mass spectra of reference D-23129 were obtained and a positive ion
+
product scan of the molecular ion (M-H) observed at m z 304 (®gure 6) revealed
}
a number of ions common to the glucuronide conjugate spectra shown in ®gure 5.
The ion at m z 304 seen in ®gure 5A and B represents the loss of the glucuronide
}

In vitro glucuronidation
437
Figure 5. Positive ion ESI LC MS product ion spectra of D-23129 glucuronides: (A) early eluting
}
}
hplc peak and (B) late eluting hplc peak.
Figure 6. Positive product ion ESI LC MS mass spectrum of D-23129.
}
}
fragment from the molecular ion. The common ions at m z 258, 230, 195 and 109
}
when compared to those observed in ®gure 6, most likely represent fragmentation of
D-23129.
Product ion scanning of the m z 478 in the negative ion mode (data not shown)
}
produced a set of ions suggesting that loss of glucuronide from the negatively

438
P. J. McNeilly et al.
Figure 7. Glucuronidation of D-23129 in human liver microsomes. Michaelis±Menton Curves
generated by incubating D-23129 at varying concentrations in microsomes (_ , HL12 ; E , HL15)
±
m
in the presence of UDPGA (2 5 m ). Data represent the means (³SD) of three determinations.
charged molecular ion was not the major fragmentation as seen in the positive ion
spectra. This diåerence might be expected since the negative charge most likely
resides on the carboxylic acid group contained in the glucuronic acid portion of the
molecule.
±
The D-23129 glucuronide with an elution time of 11 3 min was found to be
±
unstable in pH 5 0 ammonium formate acetonitrile (78 :22) at room temperature
}
(data not shown). The degradation product, D-23129, was con®rmed by mass
spectrometry and results from hydrolysis of the glucuronide conjugate. LC MS
}
analysis of the glucuronide solution with time indicated a glucuronide hydrolysis
rate with a t of approximately 100 min. In contrast, both D-23129 glucuronides
/
" #
were stable for at least one month in the microsomal incubation mixture, pH 8 0 at
±
°
®20 C.
Non-linear regression analysis of the data shown in ®gure 7 was used to
determine the apparent Michaelis±Menten constants for D-23129 glucuronide
±
formation (hplc peak at 11 3 min) by microsomal preparations from two individual
±
(48 9 pmol min mg
liver donors. K (264 lm³41 and 131 lm ³10) and V
}
}
m
max
±
±
±
protein³2 3 and 59 9 pmol min mg protein³1 1) were determined for HL12 and
}
}
HL15 respectively.
Discussion
Early D-23129 metabolic studies focused on oxidative pathways in animals and
did not address possible phase II conjugation reactions (Thomas Kronbach,
personal communication). In this study, human liver slices were used to examine the
total metabolism of D-23129 in intact tissue. Metabolites reported for ¯upirtine
formed from the cleavage of the carbamoyl side chain followed by acetylation of the
amino group were not observed in our liver slice experiments. Microsomal

In vitro glucuronidation
439
Figure 8. Postulated metabolic scheme for D-23129.
incubations with D-23129 indicated that oxidative metabolism of this compound
was minimal. Incubations of D-23129 with human liver microsomes in the presence
of UDPGA provided evidence for D-23129 glucuronide formation and material for
identi®cation of these conjugates. Biochemical and mass spectral evidence es-
tablished that the predominant D-23129 metabolic pathway was formation of two
distinct glucuronide conjugates, as proposed in ®gure 8. Based on our observations,
further animal studies conducted by the sponsor indicated species diåerences in D-
23129 metabolism and were useful in establishing appropriate toxicology studies.
Also, our D-23129 metabolic studies in human liver tissue in vitro correctly
predicted D-23129 direct glucuronidation in man. Clinical studies initiated after our
study con®rmed the presence of glucuronidated D-23129 in the urine of patients
administered the drug (Thomas Kronbach, personal communication).
Overall, the rate of D-23129 metabolism by human liver slices was less than that
observed for the microsomal incubations. This was not unexpected since rates of
metabolism may be dependant on the way slices are incubated (Dogterom 1993,
Worboys et al. 1995). It has been reported that increasing thickness of slices
produced decreased rates of metabolism when normalized to wet slice weight
(Dogterom 1993), this too may have contributed to the low production of metabolites
in liver slices. Other factors, such as cell membrane permeability, could have played
a role in the overall amount of metabolites produced in the liver slice experiments.
Although the data we have presented con®rm the direct conjugation of D-23129
by human liver tissue, the identi®cation of which compound is the secondary or
tertiary N-glucuronide is yet to be established. A third possibility of conjugation on
the carbamate nitrogen also exists and cannot be ruled out at this time.
The V
we obtained in human liver microsomes for the glucuronidation of
max
D-23129 were in agreement with those observed for tetrazole glucuronide
formation by human liver microsomes (Huskey et al. 1993) and rat recombinant
UDP-glucuronyltransferase stably expressed in V79 cells (Huskey et al. 1994), but
were lower than those observed for primary amines such as 1-naphthylamine by
±
±
expressed human UGT1 6 and UGT1 7 proteins (Orzechowski et al. 1994) and N-
acetylbenzidine by human liver microsomes (Babu et al. 1994). The glucuronidation
rates in our study are also similar to those reported for glucuronidation of tertiary
amines such as lamotrigine (Remel and Sinz 1991, Magdalou et al. 1992) and
amitriptyline (Dahl-Puustinen and Bertilsson 1987).

440
P. J. McNeilly et al.
Apparent K determined for D-23129 glucuronidation (HL12 ¯ 264 lm and
m
HL15 ¯ 131 lm ) were lower than those reported for glucuronidation of azido-
±
thymidine (K ¯ 5 mm) (Rajaonarison et al. 1991) and lamotrigine (2 6 mm)
(Magdalou et al. 1992) by human liver microsomes and were similar to those
m
observed for glucuronidation of imipramine (K ¯ 310 lm ) and chlorpromazine
m
±
(K ¯ 93 lm) using expressed human UGT1 4 protein (Green et al. 1995).
In summary, our preclinical drug metabolism studies of D-23129 using human
m
liver microsomes and liver slices in vitro provided evidence for the direct conjugation
of this compound which was not observed for the analogous compound, ¯upirtine.
The diåerences found for in vitro metabolism of D-23129 and ¯upirtine illustrate
the necessity of knowing the complete metabolic pro®le of a new drug candidate
since substitution of a single atom in a molecule can have profound eåects on its
metabolism. Con®rmation of glucuronidation of D-23129 by man in vivo also
demonstrates the value of human liver tissue metabolism studies in vitro during the
preclinical phase of drug development.
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