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Vol. 27, Issue 5, 605-612, May 1999
Corporate Research & Development ASTA Medica Group, Meissner Strasse 191, Radebeul, Germany; and Department of Pharmacology, University of California at San Diego Cancer Center, University of California, San Diego, California (N.N., C.P.S., Q.L., R.H.T.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The metabolism of retigabine in humans and dogs is dominated by
N-glucuronidation (McNeilly et al., 1997
), whereas in
rats, a multitude of metabolites of this new anticonvulsant is observed (Hempel et al., 1999). The comparison of the in vivo and in
vitro kinetics of retigabine N-glucuronidation in these
species identified a constant ratio between retigabine and retigabine
N-glucuronide in vivo in humans and dog. An
enterohepatic circulation of retigabine in these species is likely to
be the result of reversible glucuronidation-deglucuronidation reactions. Rats did not show such a phenomenon, indicating that enterohepatic circulation of retigabine via retigabine
N-glucuronide does not occur in this species. In the
rat, 90% of retigabine N-glucuronidation is catalyzed
by UDP-glucuronosyltransferase (UGT)1A1 and UGT1A2, whereas
family 2 UGT enzymes contribute also. Of ten recombinant human UGTs,
only UGTs 1A1, 1A3, 1A4, and 1A9 catalyzed the
N-glucuronidation of retigabine. From the known substrate specificities of UGT1A4 toward lamotrigine and bilirubin and
our activity and inhibition data, we conclude that UGT1A4 is a major
retigabine N-glucuronosyl transferase in vivo and
significantly contributes to the enterohepatic cycling of the drug.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Retigabine
[N-(2-amino-4-(4-fluorobenzylamino)phenyl)carbamic acid
ethyl ester] (Fig. 1) is a new
anticonvulsant that is now in clinical phase II development. It shows
effects in many in vitro and in vivo models of epilepsy and has
multiple modes of action (Kapetanovic et al., 1995; Rostock et al.,
1996; Tober et al., 1996; Skeen et al., 1995).
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The in vitro and in vivo metabolism of retigabine in humans and dog is
dominated by glucuronidation reactions resulting in the formation of
two distinct N-glucuronides (McNeilly et al., 1997). The
N2-glucuronide (retigabine N-glucuronide, Fig.
1), where the primary amino group in the 2 position is the site of glucuronidation, exceeds by far the amount of the
N4-glucuronide, where the secondary amino group in 4 position is glucuronidated. We report here only data on the formation
or cleavage of the major retigabine N-glucuronide.
Retigabine metabolism in rats is more complex. Although retigabine
N-glucuronide is the major metabolite in rat plasma and
bile, urine contains more than 20 metabolites that are mainly produced
by acetylation, ring closure reactions, and N-dealkylation
of the fluorobenzylic side chain (Hempel et al., 1999).
The importance of retigabine N-glucuronide in vivo relative to free retigabine is unclear. We therefore determined both compounds in human blood plasma from a phase I clinical study in volunteers and compared those to data from animals. Interestingly, we found that in contrast to rats, dogs and humans show a characteristic constant ratio between their retigabine and retigabine N-glucuronide plasma levels. To understand the mechanism by which the constant ratio is maintained and to understand the role of enzymes contributing to this mechanism, we: 1) characterized the enzyme kinetics of retigabine N-glucuronide formation in liver microsomes, 2) determined the interindividual variability of retigabine N-glucuronide formation in vitro in a panel of 16 human livers, and 3) characterized UDP-glucuronosyltransferases (UGTs)1contributing to retigabine N-glucuronidation by inhibition studies and by investigations with heterologously expressed enzymes. Our results suggest that the considerable interindividual variation of retigabine N-glucuronidation in vitro does not affect the ratio between retigabine and retigabine N-glucuronide in vivo and that UGT1A4 contributes significantly to the glucuronidation of retigabine.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chemicals.
Retigabine was synthesized by ASTA Medica AG (Dresden, Germany).
[14C]retigabine (9 mCi/mmol) was synthesized by
Amersham (Buckinghamshire, UK). UDP-glucuronic acid and ascorbic acid
were purchased from Fluka AG (Buchs, Switzerland). 
-Glucuronidase
(Escherichia coli), bilirubin, and alamethicin were
purchased from Sigma (St. Louis, MO). Human UGT1A1 and UGT1A4 expressed
in lymphoblasts were obtained from NatuTec (Frankfurt, Germany).
Lamotrigine was kindly provided by Glaxo Wellcome (Research Triangle
Park, NC). All other chemicals were of analytical grade and purchased
from commercial suppliers.
Preparation of Microsomes from Animals and Humans,
Frozen livers from Wistar rats (Crl:(WJ)BR, Charles River GmbH,
Sulzfeld, Germany) and Gunn Rats (HsdBlu:GUNN-j; Harlan
Winkelmann GmbH, Detmold, Germany), which are genetically different in
family I UGTs, were used for preparation of liver microsomes. Human
liver samples were obtained from livers of kidney transplant donors (Meier et al., 1983) or from livers obtained from the University of
Greifswald, Germany. Dog liver microsomes were prepared from a male
Beagle dog (Versuchstierzucht Günter Halangk, Germany). Human,
dog, and rat liver microsomes were prepared according to the method of
Meier et al. (1983) with modifications:
80°C. For the preparation of rat liver microsomes, about 3 g of liver was homogenized in 20 ml homogenization buffer with six strokes at 1200 rpm in a Potter-Elvehjem homogenizer. The remainder of the procedure was as described above.
The microsomal protein content was estimated using the bicinchoninic
acid assay (Pierce, Rockford, IL) with BSA as standard.
Preparation of Human Recombinant UGTs.
Insect Sf9 cell extracts containing expressed UGT1A1, UGT1A7, and
UGT1A10 were prepared as described previously (Strassburg et al., 1996,
1998). The entire coding region of human UGT1A3, UGT1A4, and
UGT2B7 were cloned from total liver cDNA using specific oligonucleotides to these RNAs. The cDNAs were cloned into Invitrogens BlueBac 4 transfer vector and the proteins expressed in Sf9 cells, as
described previously (Nguyen and Tukey, 1997; C.P.S., A. Strassburg, N.N., Q.L., M.P. Manns and R.H.T., in press). Human UGT1A9 and UGT2B15
were cloned directly from a human liver cDNA and expressed in Sf9 cells
as outlined (C.P.S., A. Strassburg, N.N., Q.L., M.P. Manns and R.H.T.,
in press). The cloning of the UGT2B4 cDNA was isolated from a human
liver 
cDNA library with rabbit UGT2B13 cDNA as a probe. The insert
was recovered by digestion of the plasmid with EcoRI and
BamHI, followed by cloning of the insert into pBlueBac4. The
UGT1A6 cDNA was cloned from human liver mRNA by reverse transcription
followed by PCR using specific primers that span the coding region. The
insert was cloned into pBlueBac4. The transfer vectors were then
transfected into log phase Spodoptera frugiperda
insect cell (Sf9) monolayers as described (Nguyen and Tukey, 1997).
).
Glucuronidation Assays. The final incubation mixture had a total volume of 200 µl and contained 80 to 400 µM retigabine added in 50 µl of 20% (v/v) aqueous methanol, 10 mM ascorbic acid, 7 mM MgCl2, 100 mM Tris-HCl (pH 8.0), 2.5 mM UDP-glucuronic acid (UDPGA) and 40 µg of total protein except for rat liver microsomes, where 100 µg total protein was used. The reaction was initiated at 37°C by addition of UDPGA. The mixture was then incubated for 40 min at 37°C with gentle shaking and the reaction was stopped by a 2-min incubation at 70°C. This stopping procedure resulted in an almost complete loss of retigabine N-glucuronidation activity (>99%). The amount of retigabine N-glucuronide was decreased during this incubation by about 3%. After sedimentation of the microsomal proteins at 100,000g for 20 min, the supernatant was analyzed by high-performance liquid chromatography (HPLC), as described below.
The methanol concentration of 5% (v/v) in the assay leads to a 8% loss of catalytic activity when compared with a methanol content of 1.25% (data not shown). Activation of retigabine glucuronidation by treatment with Triton X-100, Chaps, or Tween 20 could not be observed with detergent concentrations up to 0.04% (w/v) or up to a detergent to protein ratio of 2, therefore, we did not use detergents in the reaction mixture. The biosynthesis of retigabine N-glucuronide catalyzed by human and dog liver microsomes was linear as a function of incubation time up to 40 min and as a function of protein content up to 40 µg per assay. Therefore an incubation period of 40 min and a protein content of 40 µg were chosen for subsequent experiments. Because of analogous examinations the incubations in rat liver microsomes yielded a linear range up to of 100 µg of microsomal protein and 40 min of incubation time. These conditions were used for the rat assay. The synthesis of retigabine N-glucuronide by human lymphoblast-expressed UGT1A1 was linear over time up to 80 min and up to a total protein content of 70 µg. Hence an incubation period of 80 min and a protein content of 70 µg were chosen for subsequent experiments. Because of analogous examinations the incubations with human lymphoblast-expressed UGT1A4 yielded a linear range up to of 70 µg of microsomal protein and 60 min of incubation time. These conditions were used for the UGT1A4 assay. Thus, an incubation mixture had a total volume of 200 µl and consisted of 70 µg of microsomal protein, 100 mM Tris-HCl (pH 8.0), 80 to 400 µM retigabine dissolved in methanol (5% v/v final), 2.5 mM UDPGA, 10 mM ascorbic acid, 7 mM MgCl2, and 12.5 µg/ml alamethicin. Alamethicin is an antibiotic, presumably increasing the substrate availability through pore formation in the microsomal membrane. The remainder of the procedure was performed as described above. For inhibition experiments bilirubin was added to reaction mixtures containing 360 µM retigabine as a complex with BSA (final concentration: 330 µM bilirubin, 111 µM BSA) to increase the solubility of bilirubin (limit of solubility without albumin ~30 µM). Control experiments were performed in the absence of bilirubin. Inhibition experiments with lamotrigine were carried out with reaction mixtures containing 40 µM retigabine and 4 mM lamotrigine, dissolved in dimethyl sulfoxide. The final concentration for dimethyl sulfoxide and methanol in the reaction mixture was 5% (v/v) and 0.5% (v/v), respectively.Data Analysis.
Apparent Km and
Vmax values were determined with a
nonlinear least-squares fitting program Kinetik 2.4 (Arzneimittelwerk
Dresden, Germany). The analyses were done by fitting the unweighted
data using an algorithm developed by Marquard (1963). Evaluation of the
pharmacokinetic variables of the plasma concentration-time profiles was
performed using the pharmacokinetic software TopFit 2.0 (Tanswell and
Koup, 1997) by means of noncompartmental analysis. Areas under the
plasma concentration-time curve from T0 up
to T
(AUCs) were determined by the
linear trapezoidal rule and the area from the last point to infinity
was estimated by triangulation, with the terminal rate constant
determined from the slope of the regression line.
HPLC Analysis.
HPLC analyses were carried out using a HPLC system 400 (Kontron
Instruments, Neufahrn, Germany) equipped with data system 450-MT2, HPLC
pumps 420 and 422, autosampler 465, and UV detector 432. The sample
preparation was performed with column switching HPLC techniques (Roth
et al., 1981) except for using a 10-port valve instead of two 6-port
valves. Briefly, the supernatant, after centrifugation of the
incubation mixture, was injected onto a precolumn (extraction column)
which was integrated into the HPLC instrument. Here, the sample was
concentrated and highly polar compounds, e.g., salts or proteins, were
removed by flushing with 20 mM
KH2PO4 (pH 7.2), whereas
the less polar compounds were adsorbed. Three minutes after the
injection the HPLC separation on the analytical column was started with
20 mM KH2PO4 (pH
7.2)/acetonitrile 77:23 (w/w) by switching the 10-port valve. The
mobile phase and the washing buffer were gassed for 10 min with helium.
Chromatographic conditions are summarized in Table
1.
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-glucuronidase
treatment (Fig. 2). No peak at this
retention time could be detected in microsomal incubations where UDPGA
was omitted.
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Pharmacokinetic Studies in Humans. Human blood plasma samples were obtained from a clinical phase I study performed at the Department of Clinical Pharmacology, ASTA Medica AG, Frankfurt. The study was conducted in healthy male Caucasian volunteers between 18 and 45 years old. Retigabine was given over 28 days as capsules in a dosage of 2 × 200 mg per day in the morning and in the evening at least 1/2 h before a meal. On the first day only the morning dose was given. On the first day blood samples were obtained predose and at 0.33, 0.66, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 16, and 24 h. On days 3, 4, 5, 6, 7, 14, 21, 26, 27, and 28 blood sampling was taken in the morning before the next drug intake.
Additionally the blood plasma of two male healthy volunteers who received a single dose of 600 mg retigabine in capsules was analyzed. Blood samples were taken predose as well as at 0.33, 0.66, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48, and 72 h after drug administration. Samples of blood were collected using lithium heparin in Sarstedt Monovette plastic tubes and mixed gently. The tubes were then centrifuged at 3400 rpm for 10 min at 4°C. Plasma was removed and stored in polypropylene tubes at20°C until HPLC analysis. The
results from the phase I study will be reported elsewhere.
Pharmacokinetic Studies in Animals.
The pharmacokinetic studies in Beagle dogs after oral dosing of 8.25 mg/kg [14C]retigabine were performed as
described (Hempel et al., 1999). Plasma samples of rats were taken from
a pharmacokinetic study in Wistar rats after a single oral dose of 8.25 mg/kg [14C]retigabine according to the protocol
described by Hempel et al. (1999).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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HPLC Analysis of Retigabine and Retigabine N-Glucuronide. To identify the formation of retigabine N-glucuronide in vitro in microsomal incubates and in vivo in plasma and urine we developed an HPLC method for the simultaneous determination of retigabine and retigabine N-glucuronide (Fig. 3). Sample preparation was integrated into the isocratic HPLC method using a precolumn enrichment/clean-up method that yields clean samples in combination with a >98% recovery for both retigabine and retigabine N-glucuronide. The detection was linear up to 1.6 nmol for retigabine and 1.2 nmol for retigabine N-glucuronide, respectively. The limit of detection was 0.15 nmol (retigabine) and 0.05 nmol (retigabine N-glucuronide) based on a signal-to-noise ratio of 1:3.
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-glucuronidase. The sum of the peak areas of retigabine and
retigabine N-glucuronide were nearly constant over all
points. This is evidence that the extinction coefficients of retigabine and its N-glucuronide are similar. This enabled us to
determine the concentration of retigabine N-glucuronide
using the response factor of retigabine as described in Materials
and Methods.
Humans and Dogs, but Not Rats, Show a Constant Ratio between Retigabine and Its N-Glucuronide In Vivo. When we determined the concentration of retigabine and retigabine N-glucuronide in plasma of a human volunteer, we found rapid and extensive N-glucuronide formation as early as 20 min after dosing (Fig. 4). Retigabine and retigabine N-glucuronide reached the maximum plasma concentration at 40 min and 1.5 h, respectively. N-glucuronide concentrations exceeded those of retigabine by a factor of 24 ± 10-fold. In accord with this, the AUC was 24-fold larger for retigabine N-glucuronide than for retigabine. The parallel decline of plasma levels that we observed in this volunteer and two other volunteers at a higher dose (Table 2) suggested to us that a constant ratio exists between retigabine and its N-glucuronide. This is also suggested by the very similar terminal half-lives of 11.4 and 12.3 h for retigabine and retigabine N-glucuronide, respectively. This factor was slightly higher (35 ± 5) when retigabine was given to the same volunteer in a multiple dose study over another 27 days. To investigate whether this ratio is invariant in different volunteers, we determined plasma concentrations of retigabine and retigabine N-glucuronide of ten volunteers after multiple dosing on six different days. As shown in Fig. 5 the individual data points lie close to a straight line. This indicates that the ratio of retigabine N-glucuronide to retigabine is constant in different individuals (p < .001).
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), its concentration can be
approximated as the difference between total radioactivity and
retigabine concentration. The terminal half lives of retigabine as well
as of retigabine N-glucuronide were determined to be
approximately 5 h. The calculated AUC for retigabine
N-glucuronide was 25 ± 12 (n = 12)-fold higher than the corresponding value for retigabine.
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Michaelis-Menten Kinetic Analysis of Retigabine N-Glucuronidation. To investigate whether the different findings in rats in vivo can be explained by the different kinetics of retigabine N-glucuronide formation in vitro we investigated retigabine glucuronidation in liver microsomal fractions from dog, rat, and humans.
In the presence of 5% (v/v) methanol the maximum solubility of retigabine was 400 µM as determined by visual inspection of the samples after a 30-min incubation at room temperature. Due to the limited water solubility of retigabine, it was not possible to achieve concentrations that approached Vmax. The apparent kinetic constants Km and Vmax for the N-glucuronidation of retigabine in human liver microsomes were 145 ± 39 µM and 1.2 ± 0.3 nmol · min
1 · mg1
protein respectively (Fig. 7). The
corresponding values for dog and rat liver microsomes are given in
Table 3. Because of the limited
solubility of retigabine the Km of 420 µM
in rat liver microsomes is only a rough estimate. These data show that
the decreased activity of rat liver microsomes is due to both decreased Vmax and increased
Km when compared to dogs or human liver
microsomes. Although dog and human liver microsomes show similar
Vmax, apparent Km is higher in humans. The enzyme
efficiency
(Vmax/Km) is
therefore decreasing from dog to human to rat (Table 3).
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Interindividual Variation of Retigabine Glucuronidation in Humans In Vitro. Figure 8 shows the interindividual variation of retigabine glucuronidation in 16 individual samples of human liver microsomes. Saturation conditions were not reached because concentrations of retigabine higher than 400 µM exceed the solubility in our incubations. We therefore used a retigabine concentration of 360 µM, which is 2.5-fold higher than the apparent Km. From the Michaelis-Menten equation, it follows that at this concentration a velocity of 70% of Vmax is obtained.
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1 · mg1
protein and the coefficient of variation was 0.41 (S.D./mean). It is
interesting to note that in vivo we also found an ~5-fold variation of plasma levels of retigabine N-glucuronide
whereas the ratio between retigabine N-glucuronide and
retigabine was unaffected. This suggests that the variable
N-glucuronidation activity that we detected in vitro is not
responsible for the variation of retigabine N-glucuronide
levels in plasma in vivo.
Retigabine Is Glucuronidated by Multiple Isoenzymes.
Retigabine was glucuronidated by liver microsomes from Gunn rats, which
are deficient in the functional expression of the family of UGT1
enzymes. The plot of N-glucuronidation activity versus
substrate concentration (Fig. 9) was
nearly linear up to the maximal retigabine concentration of 400 µM.
Hence the Km is much higher than 400 µM.
A nonlinear regression yielded an apparent Km of 1800 ± 600 µM. Thus an exact
determination of the Michaelis-Menten constant, which should be in the
millimolar range, was not possible. The glucuronidation activity in
Gunn rat liver microsomes at a retigabine concentration of 400 µM was
0.10 nmol · min1 · mg1
protein, i.e., 42% of the glucuronidation activity in liver microsomes of wild-type Wistar rats.
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). Therefore, we tested the
inhibition of retigabine N-glucuronidation by lamotrigine, which is mainly glucuronidated by UGT1A4 (Green and Tephly, 1998). In
the presence of 4 mM lamotrigine the formation of retigabine N-glucuronide is decreased to 21 ± 3% of control
values, as tested with liver microsomes of three subjects. UGT1A1 and
UGT1A4 are the two bilirubin-glucuronidating enzymes in humans.
Therefore, we tested the inhibition of retigabine glucuronidation in
human liver microsomes of four subjects. In the presence of 330 µM
bilirubin the formation of retigabine N-glucuronide is
decreased to about 75 ± 7% of control values (Fig.
10). However, in rat liver microsomes the presence of 330 µM bilirubin decreases the amount of formed retigabine N-glucuronide to about 10% in two independent
determinations of different animals (Fig. 10). Control experiments were
performed with recombinant human UGT1A1, whereas retigabine
glucuronidation was completely inhibited in the presence of 330 µM
bilirubin. The retigabine N-glucuronidation activity by
human UGT1A4 was decreased under these conditions to about 30% (data
not shown).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study we present evidence that the new anticonvulsant retigabine is extensively N-glucuronidated in humans and that the ratio of retigabine to retigabine N-glucuronide is constant in all volunteers tested. This constant ratio between retigabine and its N-glucuronide suggests that the concentrations of these two compounds are coupled via enterohepatic circulation (EHC) and glucuronidation/deglucuronidation reactions. Here we have identified components of this potential mechanism. On the one hand, efficient glucuronidation of retigabine can be attributed to microsomal UDPGA-dependent glucuronosyl transferases. On the other hand, we show that bacterial glucuronidases cleave retigabine N-glucuronide to form retigabine. Such a mechanism could be the enzymatic basis for an efficient EHC in humans where retigabine N-glucuronide is formed in the liver (or other tissues) and is cleaved subsequently in the gut. Reabsorption of the cleaved retigabine would complete the enterohepatic cycle. Because of the quantitative importance and to explain distinctive species differences of N-glucuronide formation in vivo, we have analyzed the mechanism of retigabine N-glucuronidation in more detail using in vitro human and animal experiments.
One aim of the present study was to investigate the pharmacokinetics of retigabine N-glucuronide in human blood plasma. The concentrations of retigabine N-glucuronide and the unchanged drug after oral dosing of 200 or 600 mg retigabine are shown in Fig. 4. As can be seen from the AUC ratio of retigabine N-glucuronide and retigabine, the metabolite exceeds retigabine by about 25-fold at the 200-mg dose (Table 2). The terminal half-lives for both retigabine and retigabine N-glucuronide are very similar with a T1/2 of about 12 h. Moreover, retigabine N-glucuronide concentration normalized to the unchanged drug remained nearly constant in human blood plasma of ten volunteers after multiple oral dosage of retigabine. We speculate that the ratio between the two compounds is regulated through an EHC of retigabine in humans. The pharmacokinetic parameters from dogs for retigabine and its N-glucuronide were similar to our findings in humans (see Results, Fig. 6).
A parallel decay of the unchanged drug and its N-glucuronide
in a semilogarithmic plot was also reported for lorazepam in ponies
(Greenblatt and Engelking, 1988) and for lamotrigine in guinea pigs
(Remmel and Sinz, 1991). This pharmacokinetic behavior is probably due
to an EHC. An EHC was also described for lorazepam glucuronide in
humans (Herman et al., 1989) and acetaminophen glucuronide in rats
(Watari et al., 1984). Siegers et al. (1983) reported the EHC of
paracetamol and its glucuronide and sulfate conjugates.
The similarity of pharmacokinetics of retigabine and its
N-glucuronide in humans and dogs and the dissimilarity to
the findings in rats led us to study the kinetic data for in vitro
glucuronidation of retigabine in dog, rat, and human liver microsomes
(Table 3). The apparent Km for retigabine
glucuronidation in human liver microsomes was similar to the value
obtained by McNeilly et al. (1997). We attribute the lower
Vmax determined by McNeilly to a difference
in the experimental protocol. The apparent
Vmax value of 1.2 ± 0.3 nmol · min1 · mg1
was determined as the mean ± S.D. of the four human liver samples HGW10, HGW11, HGW11, and HGW12. Relative to the population of Fig. 8,
these samples have relatively high Vmax
values. To determine the extent of interindividual variation in the
human glucuronidation in vitro, microsomes from 16 human livers were
analyzed for retigabine N-glucuronide formation. We found a
5-fold difference in the glucuronidation activity between the different
livers. This corresponds to the in vivo observed variation of
retigabine N-glucuronide concentrations in human blood
plasma of ten different subjects (Fig. 5).
Because of the importance of the glucuronidation reaction for the
metabolic fate of retigabine, we asked whether isoforms of UGTs that
contribute to retigabine glucuronidation could be identified. The
determination of the UGT isoforms that glucuronidate retigabine in rat
and humans was approached in several ways: 1) we used expressed human
UGT isoenzymes, 2) we inhibited retigabine glucuronidation by bilirubin
and lamotrigine, and 3) we used genetically deficient Gunn rats (Owens
and Ritter, 1992). We show that among ten different human recombinant
UGT isoenzymes, UGT1A1, UGT1A3, UGT1A4, and UGT1A9 glucuronidated
retigabine. Lamotrigine, which is mainly glucuronidated by UGT1A4
(Magdalou et al., 1992; Green and Tephly, 1998), inhibited the in vitro
retigabine N-glucuronidation in human liver microsomes by
about 80%. It is therefore likely that a major part of retigabine
N-glucuronide is formed by this UGT isoenzyme. In humans,
two isoenzymes, UGT1A1 and UGT1A4, are forming bilirubin glucuronides.
Among these, UGT1A4 plays only a minor role as shown by a mRNA ratio of
5:2 (Ritter et al., 1992). Formation of retigabine
N-glucuronidation was inhibited by only 25 ± 7% of
control activity in the presence of 330 µM bilirubin. Because we
identified UGT1A3 and UGT1A9 as retigabine N-glucuronidating enzymes, it is likely that these enzymes contribute to the residual activity.
In rat liver microsomes retigabine N-glucuronidation can be
inhibited by 90% in the presence of 330 µM bilirubin. Bilirubin glucuronidation in rats is catalyzed mainly by UGT1A1 and to a lower
extent by UGT1A2. The Km value for
bilirubin-glucuronidation in rat liver microsomes is 0.9 µM
(Vanstapel and Blanckaert, 1987). Hence, in rat liver microsomes the
bilirubin glucuronidating enzymes UGT1A1 and UGT1A2 possess a greater
importance for retigabine glucuronidation when compared with humans.
These findings were further confirmed with results from glucuronidation
experiments with Gunn rat liver microsomes. The Gunn rat does not
express functional isoenzymes of UGT family 1 and therefore completely
lacks glucuronidation activity toward bilirubin and digitoxigenin
monodigitoxoside (Owens and Ritter, 1992). Our finding that retigabine
N-glucuronide is formed in Gunn rat liver microsomes with
approximately 40% of the activity of Wistar rat liver microsomes
indicates that family 2 isoenzymes contribute to retigabine
N-glucuronidation in rats.
From a clinical point of view, the biotransformation of retigabine predominantly by glucuronidation is considered to be favorable to the future treatment of patients.
First, glucuronidation is a robust metabolic step preserved even in
advanced stages of liver diseases (Hoyumpa and Schenker, 1991). Second,
the isoenzymes of glucuronyltransferases are known to have a wide
substrate specificity and the biotransformation of exogenous compounds
is usually not related to one specific enzyme. This is also true for
retigabine. Third, we have found a constant ratio between retigabine
and its N-glucuronide in vivo in different volunteers. The
observed glucuronidation variability in vitro is consequently not
relevant for the plasma clearance of retigabine.
Therefore, it is unlikely that the presence of hepatic impairment or
genetic lesions (Ritter et al., 1993) associated with a reduced hepatic
glucuronidating activity of one isoenzyme (e.g., Gilbert's syndrome,
Crigler-Najar syndrome type II) may have substantial impact on
retigabine clearance.
In summary we have shown that the constant ratio between retigabine and retigabine N-glucuronide in vivo is likely to be the result of an EHC that is mechanistically based on reversible glucuronidation-deglucuronidation reactions in humans and dog. This mechanism probably prolongs the terminal half-life of retigabine in the plasma of dogs and humans. Retigabine N-glucuronidation is mainly catalyzed by UGT1A4 in humans. Additionally, human UGT1A1, UGT1A3, and UGT1A9 also contribute to retigabine N-glucuronidation. In the rat most of retigabine N-glucuronidation is catalyzed by UGT1A1 and UGT1A2, whereas family 2 UGT enzymes also contribute to the metabolism of retigabine.
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Acknowledgments |
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We thank U.A. Meyer, Biocenter of the University of Basel, Switzerland and W. Siegmund, Institute of Clinical Pharmacology, University of Greifswald, Germany, for the supply of human liver tissue.
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Footnotes |
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Received July 22, 1998; accepted January 25, 1999.
This work was supported in part by grants from the Sächsisches Ministerium für Arbeit und Wirtschaft (project number 1791) and by United States Public Health Service Grant GM49135 (R.H.T).
Send reprint requests to: Dr. Thomas Kronbach, Corporate
Research & Development ASTA Medica Group, Biochemistry Dresden,
Arzneimittelwerk Dresden GmbH, Meiner Str. 191, D-01445 Radebeul,
Germany. E-mail: Dr_Thomas.Kronbach{at}astamedica.de
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Abbreviations |
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Abbreviations used are:
UGT, UDP-glucuronosyltransferase;
AUC, area under the plasma
concentration-time curve from t0 up to t;
HPLC, high-performance liquid chromatography;
EHC, enterohepatic
circulation;
UDPGA, UDP-glucuronic acid.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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: retigabine; 
: retigabine
N-glucuronide; 
: half of the total peak area.
