Studies on induction of lamotrigine metabolism in transgenicUGT1mice

A transgenic 'knock-in' mouse model expressing a human UGT1 locus (Tg-UGT1) was recently developed and validated. Although these animals express mouse UGT1A proteins, UGT1A4 is a pseudo-gene in mice. Therefore, Tg-UGT1 mice serve as a 'humanized' UGT1A4 animal model. Lamotrigine (LTG) is primarily metabolized to its N-glucuronide (LTGG) by hUGT1A4. This investigation aimed at examining the impact of pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor (PPAR) activators on LTG glucuronidation in vivo and in vitro. Tg-UGT1 mice were administered the inducers phenobarbital (CAR), pregnenolone-16α-carbonitrile (PXR), WY-14643 (PPAR-α), ciglitazone (PPAR-γ), or L-165041 (PPAR-β), once daily for 3 or 4 days. Thereafter, LTG was administered orally and blood samples were collected over 24 h. LTG was measured in blood and formation of LTGG was measured in pooled microsomes made from the livers of treated animals. A three-fold increase in in vivo LTG clearance was seen after phenobarbital administration. In microsomes prepared from phenobarbital-treated Tg-UGT1 animals, 13-fold higher CL_int (V_max/K_m) value was observed as compared with the untreated transgenic mice. A trend toward induction of catalytic activity in vitro and in vivo was also observed following pregnenolone-16α-carbonitrile and WY-14643 treatment. This study demonstrates the successful application of Tg-UGT1 mice as a novel tool to study the impact of induction and regulation on metabolism of UGT1A4 substrates.

Keywords: Lamotrigine; transgenic mice; uridine glucuronosyl transferases (UGTs); induction; N-glucuronide

UDP-glucuronosyltransferases (UGTs) carry out the addition of glucuronic acid (β-D-glucopyranosiduronic acid) from uridine diphosphoglucuronic acid (UDPGA) to various functional groups in xenobiotics and -endogenous compounds, thus forming the corresponding β-D-glucopyranosides (glucuronides). Glucuronides are more polar, ionizable at physiological pH and higher in molecular weight as compared with their parent drugs. This facilitates their renal as well as biliary excretion (Dutton [13]; Remmel et al. [36], [37]). The UGT superfamily of proteins consists of three families, UGT1A and UGT2B being the most important. To date 19 human UGTs have been characterized. Members of UGT1A family are -differentially expressed in organs such as liver, kidney, intestines, etc. and catalyse the conjugation of several -planar and bulky phenols, 1˚, 2˚, 3˚ and aromatic amines, carboxylic acids, and endogenous substrates such as bilirubin, bile acids, and oestrogens (Mackenzie et al. [27]; Tukey and Strassburg [53]). UGT2B family enzymes are responsible for the -glucuronidation of several xenobiotics, bile acids, prostanoids and steroidal substrates, especially androgens (Burchell et al. [9]; Mackenzie et al. [27]; Tukey and Strassburg [53]). The UGT1A family is exceptional because the entire UGT1A gene locus encodes for nine functional human UGT1A proteins, and is located on chromosome 2 (Ritter et al. [39]).

Phenobarbital (PB) is an activator of the nuclear receptor constitutive androstane receptor (CAR) (Kawamoto et al. [23]; Sugatani et al. [48]). Several studies have postulated that PB enhances the nuclear translocation of CAR in vivo (Moore et al. [32]; Sueyoshi and Negishi [47]) via a phosphorylation mechanism, based on ablation of phenobarbital induction in hepatocytes by okadaic acid (Sidhu and Omiecinski [42]). Compared with pregnane X receptor (PXR), CAR is found in low levels in the liver, has a limited tissue distribution, and is activated by fewer xenobiotics as compared with PXR (Moore et al. [32], [31]). PXR, on the other hand, is less indiscriminate in its tissue expression and is activated by a broad range of xenobiotics, pregnenolone-16α-carbonitrile (PCN) being an activator of murine PXR and rifampin an agonist of human PXR (Kliewer et al. [24]; Kobayashi et al. [25]; Moore et al. [32]). PXR is evolutionarily related to CAR (40% homology) and is thought to play a role in regulation of metabolism, transport and excretion, perhaps as a protective mechanism in cholestasis (Moore et al. [32]). A PXR-response element was identified and localized to a 386 bp region at −3529 to −3143 in the UGT1A1 5′-regulatory region. CAR/RXRa heterodimers also exhibited binding to the same element via sharing of the same DR-3 binding region. A similar DR-3 nuclear receptor response element was also identified in the UGT1A6 promoter. Nuclear receptors such as PXR and CAR have been shown to be involved in regulation of UGT1A1 gene expression both in vitro and in vivo (Barbier et al. [5]; Sugatani et al. [48], [49]; Yueh et al. [56]).

Lamotrigine (LTG) is a triazine anti-epileptic drug and is metabolized to a quaternary ammonium glucuronide, lamotrigine-2-N-glucuronide (LTGG) (Remmel and Sinz [35]; Sinz and Remmel [44]), primarily by UGT1A4 (Green et al. [19]; Green and Tephly [20]). LTG, an anti-epileptic drug, is commonly co-administered with inducing anti-epileptic agents such as carbamazepine, phenytoin, and PB. Thus, there is clinical relevance in understanding these interactions. Lamotrigine is primarily a substrate for UGT1A4 and a weak substrate for UGT1A3, with the intrinsic clearance being at least ten-fold higher for UGT1A4 as compared with UGT1A3 (Argikar and Remmel [3]). It is also understood that UGT1A4 mRNA is higher in liver than UGT1A3. Miners and co-workers recently suggested UGT2B7 may be a low-capacity, low-Km isoform for LTG glucuronidation (Rowland et al. [40]). However, we could not validate this finding in our laboratory with a sensitive liquid chromatography-mass spectrometry (LC-MS) assay. Because LTG is exclusively cleared by N-glucuronidation in humans and UGT1A4 is a pseudo-gene in mice, it is good marker of UGT1A4 activity in vivo in the Tg-UGT1 mice.

Recently, the Tukey laboratory has successfully developed and validated a novel transgenic mouse model that expresses a bacterial artificial chromosome encoding the entire human UGT1 locus (Chen et al. [10]; Senekeo-Effenberger et al. [41]). Tg-UGT1 mice have been useful for investigating the effect of induction and regulation on human UGT1A proteins and are directly applicable to a range of studies that involve tissue-specific protein expression, pharmacokinetic analysis of probe substrates, and other such studies involving xenobiotics which cannot be performed directly in humans (Argikar et al. [1]). The present study demonstrates application of Tg-UGT1 mouse model for studying the effect of PXR, CAR and peroxisome proliferator-activated receptor (PPAR)-α, -β, and -γ activators on conjugative metabolism of LTG. The objectives of the current investigation were: (1) to compare the exposure profiles of LTG in Tg-UGT1 mice and wild type mice; (2) to compare pharmacokinetic parameters of LTG in Tg-UGT1 mice receiving different inducers: PCN (PXR activator), PB (CAR activator), WY-14643 (PPAR-α activator), ciglitazone (CGZ) (PPAR-γ activator), and L-165041 (PPAR-β activator); and (3) to study the kinetics of LTGG formation in liver microsomes obtained from control Tg-UGT1, induced Tg-UGT1 and wild-type mice.

LTG standard was obtained from Toronto Research Chemicals (North York, Ontario, Canada). LTGG standard was kindly provided by Glaxo (Upper Merion, PA, USA). High-performance liquid chromatography (HPLC)-grade formic acid was obtained from Mallinckrodt Baker, Inc. (Phillipsburg, NJ, USA). HPLC-grade ammonium acetate, magnesium chloride, ethyl acetate, methanol and methyl-t-butyl ether (MTBE) were purchased from Fischer Scientific Co. (Fair Lawn, NJ, USA), the internal standard for the GC-MS assay—-2,4-diamino-6-(4-methoxyphenyl)-1,3,5-triazine—was obtained from Aldrich (St Louis, MO, USA), N-(t-butyl dimethylsilyl)-N-methyl trifluoroacetamide (MTBSTFA) (derivatization grade), glacial acetic acid, Trisma buffer, D-saccharolactone, uridine diphosphoglucuronic acid (UDPGA), PCN, WY-14643, L-165041, β-naphthoflavone (BNF), fenofibric acid (FFA), and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich Co. (St Louis, MO, USA). PB was donated by Dr. M. Negishi. High-purity solvents used for MS—acetonitrile, ethyl acetate—were obtained from Honeywell Burdick & Jackson (Muskegon, MI, USA). Alamethicin was purchased from Fluka Gmbh (Buchs, Switzerland). Nylon membrane filters were obtained from Whatman Co. (Maidstone, UK). Fresh human hepatocytes in a collagen/matrigel overlay, serum-free supplementation pack and William's E media were obtained from CellzDirect (Austin, TX, USA).

Tg-UGT1 mice generated as previously described (Chen et al. [10]) were used in compliance with the National Institutes of Health (NIH) guidelines regarding the use of laboratory animals, as described by Senekeo-Effenberger et al. ([41]). The study design is described in Table 1. Briefly, Tg-UGT1 mice were divided into separate groups of three animals each for treatment with each activator and were matched by age and sex. This was to maximize the utilization of the generated Tg-UGT1 mice.

Table 1.  Study design for the in vivo portion of this investigation. The study consisted of groups of three animals each, matched by age, gender and weight. Each inducer group was evaluated against a vehicle control. Administration of LTG was carried out orally, 24 h after the administration of the last dose of a given inducer.

Animals (20–30 g) were provided with food and water ad libitum and were maintained under controlled temperature (23°C) and lighting (12 h light/12 h dark cycles). PCN (PXR activator), WY-14643 (PPAR-α activator), CGZ (PPAR-γ activator), and L-165041 (PPAR-β activator) were dissolved in DMSO. PB (CAR activator) was dissolved in water at a concentration of 1 g l−1. Mice in each group were administered the inducer every 24 h for 3 or 4 days. PCN (100 mg kg−1) was dosed intraperitoneally, PB (0.1% w/v) was administered in the drinking water ad libitum, and PPAR agonists WY-14643 (40 mg kg−1), CGZ (15 mg kg−1) and L-165041 (30 mg kg−1) were administered intraperitoneally (Table 1). Twenty-four hours after the last dose, the mice were given LTG (20 mg kg−1, as a solution formulated as 25% hydroxypropyl-β-cyclodextrin in water) via oral gavage in the morning. This dose, when scaled per body weight, falls within the prescribed dose range for humans. Blood samples were serially drawn by retro-orbital bleeding into heparinized capillary tubes at 1, 2, 4, 8, and 24 h after the oral bolus of LTG as per the guidelines of animal care and use committee. Blood samples (30–50 μl) were transferred to microcentrifuge tubes after recording the weight and were stored in a −80°C freezer until bioanalysis. After the last sample of blood was collected, the mice were sacrificed and livers were collected and stored at −80°C.

Equal amounts of liver tissues from each treatment group were pooled and pulverized under liquid nitrogen in a mortar. The pulverized tissue was homogenized in chilled 1.15% KCl with a Potter–Elvehjem Teflon homogenizer. The homogenate was centrifuged at 2000g for 10 min at 4°C (Sorvall swinging bucket, H1000B rotor) and the supernatant was further centrifuged at 9000g for 10 min at 4°C. The resulting supernatant was then subjected to two centrifugations at 100 000g for 60 min at 4°C each (Beckman SW-40Ti Rotor). The pellet was resuspended in buffer consisting of 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, and 1 mM phenyl methyl sulfonyl fluoride. Protein concentration was determined by the Bradford method.

LTG substrate concentrations were selected based on linearity studies with time and protein and Vmax and Km estimations in pooled human liver microsomes (Argikar and Remmel [2], [3]). Glucuronidation incubations for each LTG concentration were carried out in triplicate by modification of a procedure developed earlier in this laboratory (Chen et al. [10]; Senekeo-Effenberger et al. [41]; Sinz and Remmel [43]). In brief, microsomes (0.5 mg protein content ml−1) obtained from the liver of the transgenic mice were pre-incubated with alamethicin (approximately 100 μg mg−1 of protein) on ice. Stock solutions of LTG were prepared in DMSO and 0.1 M acetic acid with the help of sonication. The final concentration of all organic solvents in each incubation did not exceed 1% of the total volume. Trisma buffer (0.1 M, pH 7.4 at 37°C) was then added, followed by the addition of 5 mM MgCl2, 5 mM D-saccharolactone and varying concentrations of LTG. The final pH was 7.2 due to the addition of the substrate in acetic acid. However, control incubations at this pH did not reveal any change in the substrate kinetics. The above incubation mixture was pre-incubated at 37°C for 2 min. The reaction was started by addition of freshly prepared 3 mM UDPGA. The final incubation volume of 200 μl was incubated for 30 min at 37°C, 30 rpm in a waterbath (Model BS-11, Lab Companion, Seoul, Korea). Control incubations lacking UDPGA did not result in metabolite formation. An equal volume of chilled acetonitrile was used to stop the reaction, followed by centrifugation at 4500g for 5 min. The supernatant was then centrifuged through nylon spin filters at 4500g for 5 min and carried forward for LC-MS analysis.

The fresh human hepatocytes (106 cells/well in a twelve-well plate) were overlaid with Matrigel/collagen and maintained in a two-dimensional sandwich configuration for 48 h before addition of the inducer. To the regular Williams' E media (WEM) obtained from CellzDirect (Austin, TX, USA), a proprietary serum-free supplementation pack from CellzDirect containing penicillin–streptomycin was added to make up enriched Williams' E media (EWEM). Solutions of EWEM containing each inducer—100 μM (PPAR-α), 1 mM PB (CAR), 50 μM RIF (PXR), 100 μM BNF (aromatic hydrocarbon receptor, AhR) and 0.5% DMSO (control)—were prepared. DMSO was used as the vehicle control. The EWEM containing each inducer was added to the wells and the cells were incubated at 37°C in a 5% CO2-humidified incubator. The media were replenished each day during the 3-day induction period. At the beginning of day 4, the cells were incubated with EWEM containing 1.25 mM LTG. After 4 h, the media were collected and analysed for formation of LTGG by the LC-MS method described below.

To quantitate LTG concentration in mouse whole blood, a sensitive GC-MS assay was developed on an Agilent 6890 series II gas chromatograph coupled with an Agilent 5973 series mass selective detector and an Agilent 7673 autosampler. Each whole blood sample (volume determined by weight) was mixed with 1 ml of MTBE for 30 s on a vortex mixer after addition of the internal standard (IS), followed by centrifugation at 13000g for 10 min in MicroMax centrifuge (International Equipment Co., White Lake, MI, USA). The organic layer was transferred to a test tube and evaporated under nitrogen on a Zymark Turbovac evaporator (Hopkinton, MA, USA). The residue thus obtained was reconstituted in MTBSTFA and transferred to an auto-sampler vial, sealed, and heated in a multi-block heater (Labline, Melrose Park, IL, USA) at 60°C for 60 min. An aliquot was injected onto a SolGel fused silica capillary column (0.25 mm i.d. × 60 m, 0.25 μm; SGE, Ringwood, VIC, Australia). The injection port temperature was set to 250°C and the detector to 310°C. Upon injection, the oven temperature was held for 2 min at 60°C. This was followed with an increase by 9°C min−1 to 150°C and held for 5 min, followed with an increase in temperature by 5°C min−1 to 250°C where it was held steady for 10 min. Finally, the temperature was ramped up by 20°C min−1 to 300°C and held for 5 min to elute high boiling-point contaminants. Derivatized LTG (m/z 426) and IS (m/z 388) eluted at 8.9 and 7.8 min, respectively, and were detected by selected-ion monitoring after electron-impact chemical ionization. Data were collected with the help of ChemStation system (Dayton, OH, USA). The limit of quantitation for this assay was 100 ng ml−1.

An LC-MS method was developed to provide the required sensitivity for quantitation of the quaternary ammonium glucuronide, as reported (Argikar and Remmel [3]). In short, LTGG ([M]+; m/z 432.2) was monitored at an optimal spray voltage of 1.9 kV and detector voltage of 1.5 kV. A capillary temperature of 250°C, an interface temperature of 250°C, a heater block temperature of 200°C, and a nebulizing gas flow rate of 0.18 l min−1 were optimized for the assay. LTGG was eluted at 2.5 min. The separation was carried out on a Higgins Analytical Haisil C-18 column (100 mm × 2.1 mm, 5 μ) at room temperature with a gradient comprised of 20–50% acetonitrile in 20 mM ammonium acetate buffer, pH 6.67, over a period of 8 min. The percentage of organic was ramped up to 80% over the next 2 min and held for 1 min, followed by a 4-min re-equilibration step. The flow rate for the analysis was 0.3 ml min−1. Data acquisition was achieved on LCMS Solution Software® from Shimadzu, Inc. (Columbia, MD, USA). The limit of quantitation for this assay was 250 ng ml−1.

Pharmacokinetic analysis for LTG in mouse whole blood was performed on a WINNONLIN non--compartmental analysis program (Pharsight, Mountain View, CA, USA), with an extra-vascular administration model for whole blood data. Dose time was set to zero minutes and doses and exact sample times for each individual mouse (amount of LTG in mg) were entered. A linear trapezoidal method with interpolation was used for the calculation of individual pharmacokinetic parameters. The data were weighted uniformly. The data obtained from in vitro incubations were fit to Michaelis–Menten kinetic model with SigmaPlot (Systat Software, Richmond, CA, USA).

LTGG levels in circulation are low. The limited sample volume from serial sampling of the Tg-UGT1 mice afforded low sample volumes for bioanalysis. Furthermore, rate and extent of LTGG formation is minimal in wild-type mice, and hence could not be measured in whole blood. Due to these reasons, only LTG levels were measured in the whole blood obtained from the Tg-UGT1 mice. Whole blood pharmacokinetic analysis of LTG yielded results as shown in Figure 1. In the mouse group receiving PCN as an inducer, the observed oral clearance of LTG (CLobs = CL/F) was 1.5-fold higher than the control group receiving DMSO. Induction with PB resulted in three-fold higher observed clearance of LTG as compared with the control group receiving water (Figure 2). Amongst the animals receiving PPAR agonists, WY-14643 administration resulted in an increase in clearance of LTG by 1.4-fold. Administration of CGZ and L-165041 resulted in decreased clearance of LTG. Mean Cmax and Tmax values are reported in Table 2.

Graph: Figure 1. Pharmacokinetic profiles of LTG in various treatment groups after oral gavage of LTG. LTG concentrations in whole blood of Tg-UGT1 mice (three mice per group) receiving specific activators of nuclear receptors—PCN (PXR activator), PB (CAR activator), WY-14643 (PPAR-α activator), CGZ (PPAR-γ activator), and L-165041 (PPAR-β activator)—were monitored by GC-MS. Standard deviation from the mean was within 20% at each concentration.

Graph: Figure 2. Kinetic profiles of LTG in Tg-UGT1 mouse whole blood. LTG clearance in Tg-UGT1 mice (three mice per group) administered: 0.1% PB in drinking water for 4 days (♦) and water control (▪).

Table 2.  LTG whole blood clearance in Tg-UGT1 mice. DMSO was a vehicle control in Tg-UGT1 mice groups treated with activators of PCN where drinking water was a control for groups treated with PB. CL/F is represented as mean value of three animals ± standard deviation.

3 *Indicates statistical significance, p < 0.05 (Student's t-test).

In the studies performed with pooled human liver microsomes, glucuronidation of LTG was found to be linear over a range of protein concentration from 0.1 to 1.0 mg ml−1, over a period of 10–80 min (Argikar and Remmel [3]). The substrate concentrations were 1.25 mM and 0.156 mM, which fall in the linear concentration range of enzyme activity, and are approximately equal to or lower than reported Km values in human liver microsomes, as described in previous literature reports (Magdalou et al. [28]) and studies from our laboratory (Argikar and Remmel [3]). At the same substrate concentration and protein concentration of 0.5 mg ml−1, the formation of LTGG was found to be linear up to 40 min of incubation, after which a moderate loss of activity was observed. Results from these preliminary studies determined that formation of the glucuronide was linear at 0.5 mg protein ml−1 for 30 min. The in vitro data were best fit to a Michaelis–Menten model as determined by residuals and Vmax and Km values were obtained. Intrinsic clearance was calculated as:

Graph

Results are reported in Table 3. Induction of CLint was highest with PB (13-fold) as compared with the control group (Figure 2). Induction by PCN was about 3.5-fold higher in the liver microsomes as compared with the DMSO control group. All animals receiving PPAR agonists showed increase in CLint of LTG. WY-14643, CGZ, L-165041 all showed more than a two-fold increase in turnover of LTG in the respective microsomes. Similar studies carried out in the wild-type mice as a part of validation of the Tg-UGT1 mouse model resulted in minimal induction and the intrinsic clearances were much lower as compared with the transgenic mice (data not shown). Comparative CLint and CLobs from in vitro and in vivo data, respectively, are represented in Tables 2 and 3.

Table 3.  Michaelis–Menten kinetics for LTG glucuronidation in Tg-UGT1 mice liver microsomes. DMSO was used as a control vehicle for Tg-UGT1 mice groups treated with all nuclear receptor activators except PB. Drinking water was used as a control vehicle for Tg-UGT1 mice treated with PB. Consideration was given to matching the genders of animals while choosing the activators and control groups. Vmax and Km are represented as means of triplicate measurements in pooled liver microsomes (from animals within each inducer group) ± standard deviation.

4 *Indicates statistical significance, p < 0.05 (Student's t-test).

A study in fresh human hepatocytes from a single donor was conducted to observe the induction by nuclear receptor activators. Results from this experiment illustrated that activators of PXR, CAR, PPAR-α and AhR increased the formation of LTGG as a result of 72-h pretreatment. After 4 h of incubation with LTG, the formation of LTGG was increased by approximately 1.5–2.3 times in cells treated with four inducers as compared with control cells exposed to vehicle alone. Results from this experiment are illustrated in Figure 3.

Graph: Figure 3. Induction of LTG glucuronidation in fresh human hepatocytes. LTG glucuronidation activity was assessed in human hepatocytes in primary culture (Matrigel/collagen sandwich) from a single donor. Activation by inducers of hPXR (RIF), AhR (BNF), PPAR-α (FFA) and CAR (PB) was compared with control (DMSO). Bars represent the means of recorded values from two wells containing 106 cells/well.

The results of the induction studies in this 'humanized UGT1A4 transgenic mouse model' (UGT1A4 being a pseudo-gene in mice) indicate that CAR, and probably PXR and PPAR-α, up-regulate UGT1A4 expression in vivo. PPARs are transcription factors belonging to the superfamily of nuclear hormone receptors, which are activated by endogenous fatty acid ligands. Three isoforms of PPARs—PPAR-α, PPAR-β (also termed as PPAR-δ) and PPAR-γ—have been reported so far (Escriva et al. [15]; Michalik and Wahli [30]). The three isoforms are selectively activated by a range of endogenous and exogenous compounds and have varying tissue distribution. Fibrates such as clofibrate, fenofibric acid, and prinixic acid (WY-14643) are potent activators of PPAR-α (Heimburger and Palmblad [21]; Hertz and Bar-Tana [22]), which has a moderate to high expression in liver. Although PPARs have been implicated to play a role in oxidative as well as conjugative metabolism of xenobiotics through modulation of drug metabolising enzymes, little information is available on the role of PPARs in the induction and regulation of specific UGTs. PPAR-α and PPAR-γ have previously been identified to play a role in up-regulation of drug-metabolizing enzymes, especially UGTs. Barbier and co-workers also found PPAR response elements in UGT2B4 (Barbier et al. [4]) and UGT1A9 (Barbier et al. [6]). PPAR-α has been implicated in increasing the levels of UGT1A4 mRNA in human hepatocyte cultures (Richert et al. [38]) and UGT1A1 in tissue culture cell lines (Brierley et al. [8]). Our group has recently shown that PPAR-α agonists induce several UGT isoforms including UGT1A1, UGT1A3, and UGT1A4 (Senekeo-Effenberger, [41]). Results from the present study indicated that induction with the PPAR-α agonist moderately increased CLint and CLobs of LTG in vitro and in vivo in the Tg-UGT1 mice. In comparison with Tg-UGT1 receiving DMSO, the Tg-UGT1 mice receiving PPAR-α agonist WY-14643 showed a two-fold increase in microsomal CLint and a 1.5-fold increase in the in vivo CLobs. Thus, PPAR-α also appears to be involved in the regulation of UGT1A4. Tg-UGT1 mice dosed with PPAR-β and -γ agonists pointed toward a lower CLobs of LTG in vivo, but approximately a moderate two-fold higher microsomal CLint. The paradoxical trend toward decrease in CLobs of LTG in animals receiving CGZ and L-165041 cannot be explained at this time. It is known that interspecies differences exists between expression of the PPAR in rodents and humans that lead to resultant differences in peroxisome proliferation (Gonzalez et al. [18]; Tugwood et al. [51]). The results of our in vivo study demonstrate that treatment of the transgenic mice with WY-14643 results in a moderate two-fold induction of lamotrigine clearance in female mice, whereas a moderate decrease in clearance was observed with prototypical PPAR-β and -γ ligands. PPAR-γ is activated by thiazolidinediones or 'glitazones' such as piotroglitazone, rosiglitazone, and CGZ (Ott et al. [33]; Parks et al. [34]; Spiegelman [45]), and has low expression in liver, with higher expression in spleen, intestine, and adipose tissue. PPAR-β, although not activated by a specific class of ligands, is activated by chemicals such as L165041, GW0742, GW2433, etc. (Desvergne and Wahli [11]; Devchand et al. [12]; Matsusue et al. [29]).

Recent work on the UGT1A4 regulatory region has shown that hepatic nuclear factor 1a (HNF1a) is critical for the basal expression of UGT1A4 (Gardner-Stephen and Mackenzie [17]) and there are two xenobiotic response element (XRE) binding sites in the first 500 bp upstream of the UGT1A4 start site (Erichsen et al. [14]). Human UGT enzymes have previously been reported to be regulated following activation of PXR (Gardner-Stephen et al. [16]; Xie et al. [54]), CAR (Sugatani et al. [50]; Xie et al. [54]), and AhR (Bock et al. 1998; Yueh et al. [55]). Significant species and ligand-dependent variation exist for activation of PXR (Moore et al. [32]). Several studies have demonstrated that human PXR (hPXR) and mouse PXR (mPXR) are activated by exclusive ligands (Kliewer et al. [24]; Kobayashi et al. [25]; Moore et al. [32]). RIF acts as a strong activator of human and rabbit PXR where as it is a weak inducer of mouse PXR. DEX and PCN are commonly used to activate mouse and rat PXR. PB is a selective agonist of CAR in rodents (Moore et al. [32]; Sueyoshi et al. [46]). In the present study, Tg-UGT1 mice receiving PB, a CAR agonist, showed approximately a 13-fold increase in microsomal CLint and a three-fold increase in the observed LTG clearance as compared with control mice (389 ± 51 ml h−1 kg−1). Tg-UGT1 mice receiving PCN, a mPXR agonist, showed a 3.5-fold increase in microsomal CLint and a modest 1.5-fold increase in the observed LTG clearance as compared with control mice receiving DMSO (254 ± 32 ml h−1 kg−1). Mice receiving PB had a 13-fold increase in microsomal CLint and the mean velocity increased more than 130-fold with a ten-fold increase in Km compared with microsomes from vehicle control-treated mice. In comparison, in microsomes from animals receiving PCN, the mean velocity increased 38-fold and the mean Km increased ten-fold as compared with the DMSO-receiving group. Thus, PB resulted in the stronger inductive response in both liver microsomes and in vivo in relation to PCN. These data suggest that both PXR and CAR may function as modulators of hUGT1A4 in the liver and that murine PXR and CAR bind to the human response elements in the regulatory region of the hUGT1A4 gene.

Full kinetic experiments in the liver microsomes revealed that in untreated mouse microsomes obtained from Tg-UGT1 mice, LTG is glucuronidated by a low-capacity, low-Km enzyme. Upon induction, both Vmax and Km increased substantially, indicating a switch to high capacity and high-Km enzyme. In vitro experiments carried out in our laboratory suggest that hUGT1A4 has similar higher Km values (Argikar and Remmel [3]). The identity of the murine enzyme responsible for the LTG glucuronidation is not known. Miners and his co-workers recently suggested based on inhibition experiments that hUGT2B7 may be a low-capacity and a low-Km isoform for LTG glucuronidation (Rowland et al. [40]). However, we could not confirm LTG N-glucuronidation with cloned and expressed hUGT2B7 with a sensitive LC-MS assay. Even though rodent UGT1A enzymes are known to be induced after AhR (Bock et al. [7]), the induction observed by an activator of CAR in the mice may not have been due to a mouse UGT1A isoform. This is because when wild-type mice were administered PB orally, the microsomal CLint and in vivo CLobs were not substantially higher than the untreated wild-type mice, thus implying that a hUGT enzyme was induced (Tables 2 and 3). Furthermore, in the wild-type mice induced with PB, the enzyme kinetics demonstrated only a low-capacity, low-Km enzyme. Our findings demonstrate that mouse CAR is activated by PB in liver and can translocate from the cytosol and bind to CAR response elements on hUGT1A4 in the Tg-UGT1 mice (Bock et al. [7]; Koike et al. [26]). PB has been reported to be one of the most selective CAR inducers, though it is speculated that it may activate PXR at higher concentrations (Moore et al. [32]; Sueyoshi and Negishi [47]). CAR has been previously reported to play a role in modulation of hUGT1A1, the enzyme responsible for the metabolism of bilirubin, through its effect on the UGT1A1 gene (Sugatani et al. [48], [49], [50]). The increase in in vivo clearance for LTG in the PB-treated group and increase in in vitro CLint values for all treatment groups were statistically significant (p < 0.05, Student's t-test). However, the current study design is limited by the number of animals per group for further, more powerful statistical analysis among various groups treated with inducers. The current results point toward a possible induction of LTG glucuronidation in the Tg-UGT1 mice as a result of administration of nuclear receptor inducers.

Our studies with fresh human hepatocytes also illustrated an increase in turnover of LTG in cells treated with various nuclear receptor agonists (Figure 3). It must be noted that the induction experiment in fresh human hepatocytes was conducted in cells obtained from a single donor and a comprehensive study in cells from multiple donors may be conducted to validate these results further. Such a study, however, was not within the scope of the current investigation. Overall, the in vitro results point to involvement of PPAR, PXR and CAR in up--regulation of UGT1A4 enzyme in the liver. PCN, a murine PXR agonist, modulated the observed blood clearance as well as the hepatic intrinsic clearance of LTG. PB was found to be the most potent inducer and increased the blood CLobs of LTG by three-fold. It can be predicted that nuclear receptors as above could be involved in similar LTG clearance modulation in humans. For a drug with a high extraction ratio, the in vivo hepatic clearance is predominantly determined by the hepatic blood flow and may not be altered to a great extent by an alteration in CLint. However, the effects of inducing agents may be significant on clearance of drugs with lower hepatic extraction ratio like LTG, where hepatic clearance is primarily a function of CLint.

The transgenic mice used in this study are valuable for functional studies with UGT1A4 because it is a pseudo-gene in rodents. However, these mice still express a murine UGT1A locus and induction of hUGTs relies on the murine transcription factors. Further progress in eliminating the mouse UGT1A background has recently been accomplished in the Tukey laboratory (Tukey [52]). Future work with this novel in vivo model of human glucuronidation will involve expression of the human transcription factors to obtain a truly 'humanized' mouse model for xenobiotic metabolism and toxicity studies. It is clear that activators of these receptors play a critical role in modulation of hepatic and extra-hepatic metabolism through their effects on UGT1A gene locus. The identification of critical pathways involved in the induction and regulation of the UGT1A enzymes will lend an additional perspective to the understanding of conjugative metabolism of xenobiotics, and the mechanisms of drug–drug interactions involving UGTs.

This study was jointly funded by a grant from the National Institute of Neurological Disorders and Stroke NIH (Grant Number NINDS P50 NS16308) and US Public Health Service Grants (Numbers GM49135 and ES10337). The authors thank Glaxo (GSK) for providing the lamotrigine-2N-glucuronide standard and Dr. M. Nigeshi for providing phenobarbital. The authors thank Ms. Falguni Gadkari for reviewing the manuscript. The assistance of Ms. MyHang Tran with Winnonlin is greatly appreciated.

Declaration of interest: Professor Rory P. Remmel has an investigator-initiated grant from GlaxoSmithKline for a clinical study on LTG pharmacokinetics. He holds no financial interest in the company.