Lamotrigine [3,5-diamino-6-(2,3-dichlorophentenyl)-1,2,4-triazine; LTG] (Fig. 1) is an anticonvulsant with proven efficacy in the treatment of epilepsy (Pellock, 1994). It is one of a few agents approved for the treatment of both partial and generalized seizures, and is well tolerated (Vinik, 2005). Hepatic metabolism is the primary route of elimination for LTG, predominantly via N-glucuronidation (Cohen et al., 1987; Mikati et al., 1989). After an oral dose of LTG, approximately 85% is recovered in urine as a quaternary glucuronide at the N-2 position of the triazine ring (Fig. 1). The remainder is eliminated as the N5 glucuronide, an N2-methylated derivative, unidentified metabolites, and unchanged drug (Sinz and Remmel, 1991; Doig and Clare, 1991).

Glucuronidation reactions are catalyzed by the enzyme UDP-glucuronosyltransferase (UGT). UGT exists as a superfamily of enzymes that exhibit distinct, but overlapping, substrate and inhibitor selectivities (Miners et al., 2004; Kiang et al., 2005; Mackenzie et al., 2005). Seventeen human UGT proteins have been identified to date, and these have been classified into two families (UGT1 and UGT2) based on sequence identity (Mackenzie et al., 2005). Although the disposition of LTG is well characterized in vivo, the identity of the human hepatic UGT enzyme(s) responsible for the elimination of this drug has not been explored in a systematic manner. Green and Tephly (1996) reported that UGT1A4 glucuronidated LTG. In contrast UGT1A3, another enzyme known to glucuronidate amines, lacked LTG N2-glucuronidation activity. Given the broad role of UGT1A4 in amine glucuronidation, it is widely assumed that this enzyme is the principal catalyst of LTG N2-glucuronidation. However, the relative contribution of UGT1A4 to human liver microsomal LTG N2-glucuronidation at therapeutic concentrations is unknown.

Although LTG monotherapy has been shown to be effective in the treatment of epilepsy, this drug is more commonly administered in combination with other anticonvulsants (Hirsch et al., 2004). Thus, the potential exists for inhibitory drug-drug interactions. Indeed, coadministration of valproic acid (VPA), another anticonvulsant that is primarily eliminated by glucuronidation (Cotariu and Zaidman, 1988), is known to increase the area under the plasma-concentration time curve (AUC) for LTG in a dose-dependent manner in humans (Morris et al., 2000).

Recent work in this and other laboratories has explored in vitro approaches for predicting the hepatic clearance of glucuronidated drugs in vivo and the magnitude of inhibitory drug interactions involving glucuronidated drugs. In general, kinetic data generated using human liver microsomes (HLMs) as the enzyme source underestimate both hepatic clearance (CL_H) and the magnitude of in vivo interactions for glucuronidated drugs (Boase and Miners, 2002; Soars et al., 2002). It has been observed, however, that the addition of bovine serum albumin (BSA) (2%) to incubations results in an increased in vitro CL_int for zidovudine and a decreased K_i for fluconazole inhibition of zidovudine metabolism, such that the magnitude of the inhibitory interaction in vivo was predicted correctly (Uchaipichat et al., 2006b). The mechanism by which BSA decreases the K_m of zidovudine and the K_i of fluconazole is unknown but is independent of drug and inhibitor protein binding. This result is in agreement with data published for CYP2C9 substrates, which show that the addition of BSA improves the predictive capacity of models used to predict the clearance of several drugs (Ludden et al., 1997; Carlile et al., 1999; Tang et al., 2002).

There is emerging evidence that UGTs other than UGT1A4 catalyze N-glucuronidation reactions. For example, it has been demonstrated recently that UGT2B7 mediates the N-glucuronidation of carbamazepine and BMS-204352 (Staines et al., 2004; Zhang et al., 2004), and multiple UGT1A subfamily enzymes metabolize retigabine (Hiller et al., 1999). This work sought to characterize the kinetics of LTG glucuronidation by HLMs and recombinant human UGTs and to assess the contribution of known UGTs to LTG hepatic clearance. In addition, the mechanism of the LTG-VPA interaction was investigated. Studies were performed with and without added BSA to determine whether in vitro data accurately predicted the magnitude of the LTG-VPA interaction, as has been recently reported for the fluconazole-zidovudine interaction.

Materials and Methods

Materials. Alamethicin (from Trichoderma viride), BSA (fraction V, 98-99% protein), hecogenin (Hec), 4-methylumbelliferone (4MU), 4-methylumbelliferone β-d-glucuronide (4MUG), UDP-glucuronic acid (trisodium salt), and VPA were purchased from Sigma-Aldrich (Sydney, Australia). Lamotrigine (LTG) and lamotrigine N2-glucuronide (LTG-Gluc) were obtained from Wellcome Research Laboratories (Beckenham, UK). Solvents and other reagents were of analytical reagent grade.

Methods.Human Liver Microsomes and Expressed UGT Protein. Liver tissue (H07, H10, H12, H13, and H40) was obtained from the human liver “bank” of the Department of Clinical Pharmacology of Flinders University. Approval for the use of human liver tissue in xenobiotic metabolism studies was obtained from both the Clinical Investigation Committee of Flinders Medical Centre and from the donors' next-of-kin. HLMs were prepared by differential centrifugation, as described by Bowalgaha et al. (2005).

cDNAs encoding UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, 2B17, and 2B28 were stably expressed in a human embryonic kidney cell line (HEK293), as described previously (Sorich et al., 2002; Stone et al., 2003; Uchaipichat et al., 2004). Cell lines were transfected separately with UGT cDNAs cloned into the expression vector pEF-IRES-puro6. The cells were incubated in Dulbecco's modified Eagle's medium, which contained puromycin (1 mg/l), fetal calf serum (10%), and penicillin G (100 units/ml)-streptomycin (100 mg/l) in a humidified incubator at 37°C with an atmosphere of 5% CO₂. After growth to at least 80% confluence, cells were harvested and washed with phosphate-buffered saline (0.1 M, pH 7.4). Cells were subsequently lysed by sonication using a Heat Systems-Ultrasonics sonicator (John Morris Scientific, Sydney, Australia) set at a microtip limit of four. Cells expressing UGT1A enzymes were sonicated with four “bursts,” each lasting 2 s, separated by 3 min with cooling on ice. Cells expressing UGT2B enzymes were treated using the same method, except that sonication was limited to 1-s bursts. Lysed samples were centrifuged at 12,000g for 1 min at 4°C, and the supernatant fraction was removed and stored at -80°C until use. Expression of each UGT was demonstrated by immunoblotting with a commercial UGT1A antibody and a nonselective UGT antibody (raised against purified mouse Ugt) (Uchaipichat et al., 2004) and, where possible, activity measurements (see below).

Confirmation of UGT Activity. Activities of recombinant UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, 2B17, and 2B28 were confirmed using the nonselective substrate 4MU. For all enzymes except UGT2B4 and UGT2B28, the conversion of 4MU to 4MUG was measured according to the spectrofluorometric procedure of Miners et al. (1988), as modified by Uchaipichat et al. (2004). The concentration of 4MU present in incubations corresponded to the known K_m (or S₅₀) for each enzyme (Sorich et al., 2002; Uchaipichat et al., 2004). Given the lower specific activities of UGT2B4 and 2B28, 4MU glucuronidation activity (at 1 mM) was confirmed using a radiometric thin-layer chromatographic procedure (Jin et al., 1997). UGT1A4 activity was confirmed using trifluoperazine as the substrate, at the approximate K_m for this substrate (40 μM), according to the method of Uchaipichat et al. (2006a). The activity of UGT2B10 toward 4MU, and indeed, most other substrates, is low or absent (Miners et al., 2004). Thus, it was not possible to include a positive control for this enzyme.

LTG N2-Glucuronidation Assay. Incubations, in a total volume of 200 μl, contained 4 mM MgCl₂, 0.1 mg of HLMs, and 10 to 3000 μM LTG. The LTG was dissolved in 1 M phosphoric acid containing 10% acetonitrile. Phosphate buffer (0.1 M, pH 7.4) was generated in situ by the addition of KOH (1 M, 37.2 μl) to each incubation tube. LTG solutions were diluted 1:10 upon addition to incubations, such that the final concentration of acetonitrile was 1% v/v. This concentration of acetonitrile has a minor effect on UGT enzyme activities (Uchaipichat et al., 2004). Given the limited solubility of LTG, the maximal concentration possible in incubations was 3000 μM. HLMs were fully activated by the addition of the pore-forming polypeptide alamethicin (50 μg/mg protein) with incubation on ice for 30 min (Boase and Miners, 2002). After a 5-min preincubation, reactions were initiated by the addition of UDP-glucuronic acid (5 mM). Incubations were performed at 37°C in a shaking water bath for 60 min. Reactions were terminated by the addition of perchloric acid (2 μl, 70% v/v). Samples were subsequently centrifuged at 4000g for 10 min, and a 40-μl aliquot of the supernatant fraction was injected directly into the HPLC column. For reactions performed using recombinant UGTs, incubation mixtures contained HEK293 cell lysate (0.2 mg) in place of HLM protein, and the incubation time was increased to 75 min. Incubations containing BSA (2%) were terminated by the addition of perchloric acid (6 μl, 70% v/v solution). Under the reaction conditions used, LTG N2-glucuronidation was linear with respect to incubation time to 75 min and protein concentration to 1.5 mg/ml with both HLMs and HEK293 cell lysate as the enzyme source. LTG N2-glucuronidation by lysate from untransfected HEK293 cells was not detectable.

Quantification of LTG-Gluc Formation. HPLC was performed using an Agilent 1100 series instrument (Agilent Technologies, Sydney, Australia) fitted with a Zorbax Eclipse XBD-C8 analytical column (4.6 × 150 mm, 5 μm; Agilent Technologies). Analytes were separated by gradient elution at a flow rate of 1 ml/min. Initial conditions were 91% phosphate buffer (25 mM, pH 7.4), containing triethylamine (200 μl/l) (mobile phase A) and 9% acetonitrile (mobile phase B). These conditions were held for 3 min; then, the proportion of mobile phase B was increased to 18% over 4 min and held for 1 min. Finally, the proportion of mobile phase B was increased to 55% over 1 min. Column eluant was monitored by UV absorbance at 254 nm. Retention times for LTG-Gluc and LTG were 5.3 and 10.3 min, respectively. LTG-Gluc formation was quantified by comparison of peak areas to those of an authentic LTG-Gluc standard curve prepared over the concentration range 1 to 20 μM. The lower limit of quantification, defined as 5 times background absorbance, was 4 pmol/LTG-Gluc injected (corresponding to a rate of approximately 1 pmol/min · mg using recombinant UGTs as the enzyme source). Overall within-day assay reproducibility was assessed by measuring LTG-Gluc formation in eight separate incubations of the same batch of HLMs (H40). Coefficients of variation were 4.1% and 2.4% for LTG concentrations of 100 μM and 3000 μM, respectively.

Inhibition of LTG-Gluc Formation. Incubations of both HLMs and recombinant UGTs were performed in the absence and presence of Hec and VPA. Hec (10 μM), a known highly selective inhibitor of UGT1A4 (Uchaipichat et al., 2006a), was added to incubations to assess the contribution of this enzyme to LTG glucuronidation. VPA inhibition of LTG glucuronidation was investigated using microsomes from five human livers (H7, H10, H12, H13, and H40) and with UGT1A4 HEK293 cell lysate. Experiments performed to determine the inhibitor constant (K_i) for VPA inhibition of LTG glucuronidation included four VPA concentrations (300, 600, 900, and 1200 μM) at each of three LTG concentrations (200, 400, and 600 μM).

Binding of LTG and VPA to BSA, HLMs, and HEK293 Cell Lysate. Binding of LTG and VPA to HLMs, HEK293 cell lysate, BSA (2%), and mixtures of BSA with each enzyme source was measured using an equilibrium dialysis method (McLure et al., 2000). Binding measurements were performed using a Dianorm equilibrium dialysis apparatus (Dianorm, Munich, Germany) that comprised Teflon dialysis cells (capacity of 1200 μl per side) separated into two compartments with Sigma-Aldrich dialysis membrane (molecular mass cutoff 12 kDa). Membranes were conditioned overnight at 4°C in phosphate buffer (0.1 M, pH 7.4). One side of the dialysis cell was loaded with 1 ml of a solution of either LTG (12.5-3000 μM) or VPA (300-1200 μM) in phosphate buffer (0.1 M, pH 7.4). The other compartment was loaded with 1 ml of either a suspension of HLMs (0.5 mg) in phosphate buffer (0.1 M, pH 7.4), HEK293 cell lysate (1 mg) in phosphate buffer (0.1 M, pH 7.4), BSA (2%) in phosphate buffer (0.1 M pH 7.4), or a combination of BSA (2%) with each enzyme source in phosphate buffer (0.1 M, pH 7.4). The dialysis cell assembly was immersed in a water bath maintained at 37°C and rotated at 12 rpm for 5 h. Control experiments were also performed with phosphate buffer or HLMs (0.25 mg) on both sides of the dialysis cells at low and high concentrations of both drugs to ensure that equilibrium was attained. A 200-μl aliquot was collected from each compartment, treated with ice-cold methanol containing 4% glacial acetic acid (200 μl), and cooled on ice. Samples were subsequently centrifuged at 5000g for 10 min at 10°C, and an aliquot of the supernatant fraction (5 μl for LTG, 40 μl for VPA) was analyzed by HPLC.

Quantification of LTG and VPA Binding. The HPLC system used was as described previously for LTG-Gluc formation. Separation of LTG was achieved using a 63:37 mixture of mobile phases A and B used for the LTG-Gluc assay. The mobile phase flow rate was 1.0 ml/min and column temperature was 25°C. Column eluant was monitored at 254 nm. The retention time for LTG was 2.6 min. VPA separation was achieved using an isocratic mobile phase comprising a 55:45 mixture of phosphate buffer (pH 3.1, 25 mM) and acetonitrile at a flow rate of 1.0 ml/min and a column temperature of 25°C. Column eluant was monitored at 210 nm. The retention time for VPA was 2.9 min. LTG and VPA concentrations in dialysis samples were determined by comparison of peak areas to those of a standard curve, in the respective concentration ranges 50 to 3000 μM and 300 to 1200 μM. Within-day variation was assessed by measuring LTG (50 and 1500 μM) or VPA (300 and 1200 μM) (n = 5 for each concentration) content in samples containing phosphate buffer (0.1 M, pH 7.4), HLMs (0.5 mg) in phosphate buffer (0.1 M, pH 7.4) or a mixture of BSA with HLMs in phosphate buffer (0.1 M, pH 7.4). Coefficients of variation were less than 5% in all cases.

Data Analysis. Kinetic constants for LTG glucuronidation by recombinant UGT1A4 were generated by fitting experimental data to the Michaelis-Menten equation, whereas kinetic constants for LTG glucuronidation by HLMs were obtained by fitting data to a hybrid Michaelis-Menten-Hill equation using EnzFitter (Biosoft, Cambridge, UK): where [S] is the substrate concentration, K_m is the Michaelis constant (substrate concentration at 0.5 V_max), S₅₀ is the concentration at 0.5 V_max, n is the Hill coefficient (which reflects the degree of sigmoidicity of the velocity versus substrate-concentration relationship), and V_max1 and V_max2 are the maximal velocities for the two enzymes.

K_i values for VPA inhibition of LTG glucuronidation were determined by fitting experimental data to the expression for the competitive, noncompetitive, uncompetitive and mixed inhibition models using EnzFitter (Biosoft). Goodness of fit to kinetic and inhibition models was assessed by comparison of the F-statistic, r² values, parameter standard errors, and 95% confidence intervals. Kinetic constants are reported as the mean value ± standard deviation of the parameter estimate.

Statistical Analysis. Where appropriate, statistical analysis (Wilcoxon signed rank test) was performed using SPSS 10.0.5 (SPSS Inc., Chicago, IL). Values of p less than 0.05 were considered significant.

Quantitative Prediction of the VPA-LTG Interaction. The magnitude of VPA inhibition of LTG hepatic clearance was predicted based on the increase in the AUC ratio caused by the presence of the inhibitor, using the equation: where K_i is the inhibition constant determined in vitro, f_h is the ratio of hepatic to total clearance expressed as a fraction, f_m is the ratio of metabolic clearance to total clearance expressed as a fraction, and [I] is the estimate of in vivo inhibitor concentration (Ito et al., 1998).

Different values of [I] were used to predict the magnitude of the interaction between LTG and VPA, based on approaches described by Ito et al. (1998, 2004). These were: maximum inhibitor concentration in plasma ([I]_max), maximum unbound inhibitor concentration in plasma ([I]_max.unbound), average inhibitor concentration in plasma ([I]_ave), average unbound inhibitor concentration in plasma ([I]_ave.unbound), and average and maximum hepatic input concentration given by the inhibitor concentration in the blood plus the inhibitor concentration from gastrointestinal absorption ([I]_inlet). The latter parameter was calculated as: where [I]_max is the maximum inhibitor concentration in blood, k_a is the absorption rate constant, f_a is the fraction of the inhibitor dose absorbed from the gastrointestinal tract, and Q_H is liver blood flow. The dose was taken as 500 mg (3467 μmol), assuming that an “average” daily dose is 500 mg twice daily. It should be noted that increasing the dose to 1000 mg resulted in approximately a 9% increase in the AUC ratio (see Table 3) determined using the hepatic input concentration. [I]_max was taken as the upper limit of the plasma VPA therapeutic range for the treatment of epilepsy; that is, 100 mg/l (693 μM) (Dutta et al., 2003). The lower limit of the VPA therapeutic range is 50 mg/l (347 μM). Thus, the mid-point of the therapeutic range (520 μM) was used as [I]_ave. The unbound values of [I]_inlet.max and [I]_inlet.ave were calculated by multiplying the respective in vivo concentrations by the fraction of VPA unbound in blood (f_u(VPA)), which was taken as 0.1 (Anderson et al., 1994). Because VPA is essentially completely absorbed from the gastrointestinal tract, f_a is 1.0 (Bressolle et al., 1994). The absorption rate constant (k_a) for VPA was assumed to be 0.1/h (Ito et al., 2004). LTG AUC data for patients administered different doses of VPA have been reported by Morris et al. (2000). A VPA dose of 1000 mg/day increased the LTG AUC by 160%. Thus, the in vivo LTG AUC ratio (AUC_(i)/AUC) was taken as 2.60.

Results

Binding of LTG and VPA to HLMs, HEK293 Cell Lysate and BSA. The binding of LTG and VPA was calculated as the concentration of drug in the buffer compartment divided by the concentration of drug in the protein compartment, and was expressed as the fraction unbound in incubations (f_u,inc). LTG and VPA binding to both HLMs and HEK293 cell lysate was negligible (<5%) across the concentration ranges investigated. Both drugs did, however, exhibit significant binding to BSA (2%). The binding of LTG to BSA was independent of LTG concentration, but VPA f_u,inc increased with increasing VPA concentration. The mean (±S.D.) f_u,inc value for LTG binding to BSA was 0.65 ± 0.03, whereas for VPA, f_u,inc ranged from 0.15 ± 0.04 (at 300 μM) to 0.35 ± 0.02 (at 1200 μM). Significant binding was also observed for both drugs with mixtures of BSA (2%) and each protein source. Mean f_u,inc values for LTG binding to mixtures of BSA and HLMs and to BSA and HEK293 cell lysate were 0.49 ± 0.07 and 0.30 ± 0.01, respectively. Binding was independent of LTG concentration and, notably, was not additive with the binding of LTG to each source individually. In contrast, mean f_u values for binding of VPA to mixtures of BSA and HLMs and BSA and HEK293 cell lysate were identical to those for binding to BSA alone, ranging from 0.15 ± 0.01 (at 300 μM) to 0.35 ± 0.03 (at 1200 μM). It was also demonstrated for both LTG and VPA that the presence of the alternate drug had no effect on binding under the conditions investigated. Where binding was observed (to HLMs or BSA), the concentration of drug added to incubation mixtures was corrected for binding in calculations of kinetic parameters.

Kinetics of LTG N2-Glucuronidation by HLMs. Although data were adequately fitted to the two-enzyme Michaelis-Menten equation, LTG N2-glucuronidation by HLMs in the presence and absence of BSA was best described by a hybrid model comprising the Michaelis-Menten and Hill (with n <1, i.e., negative cooperativity) equations (Fig. 2); all goodness of fit parameters (see Data Analysis) were superior using the hybrid model. Derived kinetic parameters are shown in Table 1. In the absence of BSA, the mean K_m value of the Michaelis-Menten component was 2234 ± 774 μM, whereas the mean S₅₀ and n values of the Hill component were 1869 ± 1286 μM and 0.65 ± 0.16, respectively. The addition of BSA did not significantly affect the K_m but did cause a significant (p < 0.05) decrease in the mean S₅₀ (by approximately 65%) (Table 1). In the absence of BSA, the mean (±S.D.) V_max values for the Michaelis-Menten and Hill components were 594 ± 333 and 162 ± 108 pmol/min · mg, respectively. BSA did not have a significant effect on the mean V_max of either component, nor did it affect the Hill coefficient. It should be noted, however, that because the maximum unbound concentration of LTG in incubations conducted in the presence of BSA was approximately 1500 μM (i.e., maximum total concentration 3000 μM with f_u 0.49), kinetic constants obtained from the experiments in the presence of albumin should be considered as estimates (although data for the Michaelis-Menten component are internally consistent).

Kinetics of LTG N2-Glucuronidation by HLMs in the Presence of Hecogenin. The highly selective UGT1A4 inhibitor Hec (Uchaipichat et al., 2006a) was added to incubation samples (final concentration 10 μM) to confirm the involvement of this enzyme in human liver microsomal LTG N2-glucuronidation. Derived kinetic parameters are shown for experiments performed in the presence and absence of BSA (2%) in Table 2. LTG-Gluc formation by HLMs in the presence of Hec, with and without 2% BSA, was described by the Hill equation with n <1 (Fig. 2); that is, Hec abolished the Michaelis-Menten component of LTG N2-glucuronidation. In the absence of BSA, the mean (±S.D.) value of S₅₀ was 1848 ± 256 μM. For all livers, the addition of 2% BSA to incubation samples caused a significant (p < 0.05) reduction in the S₅₀ (by approximately 80%), and a relatively minor, but significant (p < 0.05), reduction in mean V_max (approximately 30%). The addition of 2% BSA had no effect on the Hill coefficient.

LTG N2-Glucuronidation by Recombinant UGTs. Activities of UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 with 4MU as the substrate were similar to those reported by Uchaipichat et al. (2004). 4MU glucuronidation activity of UGT 2B4 and 2B28 was confirmed using a radiometric thin-layer chromatographic method. UGT1A4 activity with trifluoperazine as the substrate was in agreement with previously reported data (Uchaipichat et al., 2006a). Together with UGT2B10 and 2B28, which show very low or no activity toward all substrates tested to date, these enzymes were screened for LTG N2-glucuronidation activity. At an LTG concentration of 1500 μM, UGT1A4 exhibited the highest activity (105 p mol/min · mg), whereas UGT1A1, UGT1A3, UGT1A6, UGT1A7, and UGT2B7 exhibited lesser activity (<6% that of UGT1A4).

Kinetics of LTG N2-Glucuronidation by Recombinant UGT1A4. LTG-Gluc formation by UGT1A4, in the presence and absence of 2% BSA, exhibited Michaelis-Menten kinetics (Fig. 3). In the absence of BSA, the K_m and V_max values for LTG-Gluc formation by UGT1A4 were 1558 μM and 224 pmol/min · mg, respectively. The addition of 2% BSA resulted in significant (p < 0.05) increases in both K_m and V_max (to 3235 μM and 1278 pmol/min · mg, respectively). As with previous experiments conducted in the presence of BSA, the maximum unbound concentration of LTG in incubations was approximately 1500 μM, and hence derived kinetic parameters should be considered as estimates. The addition of Hec (10 μM) to incubations performed with recombinant UGT1A4 decreased the rate of LTG-Gluc formation at substrate concentrations ranging from 10 to 3000 μM by approximately 95% (data not shown). The low rate of formation of LTG-Gluc by UGT1A1, 1A3, 1A6, 1A7, and 2B7 HEK293 cell lysates precluded full kinetic analysis.

Inhibition of LTG-Gluc Formation by UGT2B7 Substrates/Inhibitors. The known UGT2B7 substrate zidovudine (3 mM) (Court et al., 2003) and the selective UGT2B7 inhibitor fluconazole (2.5 mM) (Uchaipichat et al., 2006b) inhibited LTG-Gluc formation by pooled HLMs at an LTG concentration of 25 μM by 70% and 74%, respectively, and by 22% and 20%, respectively at an LTG concentration of 1500 μM (data not shown).

VPA Inhibition of LTG-Gluc Formation Catalyzed by HLMs and Recombinant UGT1A4. Inhibition of UGT1A4-catalyzed LTG N2-glucuronidation by VPA was investigated in the presence and absence of 2% BSA. VPA caused <5% inhibition of LTG-Gluc formation by recombinant UGT1A4, irrespective of the presence of BSA. Moreover, VPA (10 mM) added to incubations of pooled HLMs essentially abolished the Hill (i.e., non-UGT1A4) component of LTG N2-glucuronidation over the substrate concentration range 25 to 3000 μM. Kinetic data generated in the presence of VPA (10 mM) were well described by the Michaelis-Menten equation (data not shown); derived K_m (1309 μM) and V_max (686 pmol/min · mg) were within the ranges obtained for the Michaelis-Menten component (i.e., UGT1A4) of the LTG N2-glucuronidation by microsomes from individual livers in the absence of BSA (Table 1). Taken together, these observations indicate that VPA has no effect on UGT1A4-catalyzed LTG N2-glucuronidation. Hec (10 μM) was therefore added to incubations to remove the UGT1A4 component of the microsomal reaction. For incubations conducted in the presence of Hec, VPA inhibited LTG N2-glucuronidation by HLMs in a competitive manner in the presence and absence of BSA (Fig. 4). The addition of 2% BSA to incubation samples caused a significant (p < 0.05) reduction in the mean K_i, determined with microsomes from livers H7, H10, H12, H13, and H40, from 2465 ± 370 to 387 ± 12 μM. The addition of 2% BSA did not alter the model (i.e., competitive) of inhibition. As noted previously, correction of the binding of LTG and VPA to BSA (and other incubation components) was accounted for in the calculation of K_i values.

Prediction of in Vivo AUC Ratio. Predicted LTG AUC ratios in vivo (in the presence and absence of coadministered VPA) based on various value of [I] (see Materials and Methods) are shown in Table 3 for K_i values generated in both the absence and presence of BSA. Predicted AUC ratios calculated using K_i values generated in the absence of BSA ranged from 1.00 to 1.28. The use of K_i values generated in the presence of BSA resulted in higher predicted AUC ratios (1.01-2.31). However, only calculations that incorporated the total VPA (inhibitor) concentration predicted a clinically significant interaction (predicted AUC ratio 1.86-2.31).

Discussion

The microsomal kinetic studies reported here are consistent with the involvement of at least two UGTs in LTG N2-glucuronidation. Kinetic data were well described by a hybrid model, comprising Michaelis-Menten and Hill components. Hec, a highly selective inhibitor of UGT1A4 (Uchaipichat et al., 2006a), abolished activity of the Michaelis-Menten component without affecting the kinetic parameters for the Hill component. The mean K_m for the Michaelis-Menten component of LTG N2-glucuronidation by HLMs in the absence of Hec (viz. 2234 ± 774 μM) was similar in value to the K_m for UGT1A4-catalyzed LTG N2-glucuronidation (1558 μM). Taken together, these data indicate that UGT1A4 is responsible for the Michaelis-Menten component of the microsomal reaction.

Apart from recombinant UGT1A4, UGT 1A1, 1A3, 1A6, 1A7, and 2B7 catalyzed LTG N2-glucuronidation, albeit at lower rates (3.1-5.4 pmol/min · mg versus 105 pmol/min · mg at a substrate concentration of 1500 μM). The low activities of these enzymes precluded full kinetic analysis. Substitution of the mean kinetic parameters for human liver microsomal LTG N2-glucuronidation in the hybrid Michaelis-Menten-Hill equation indicated that the contribution of the Hill component to the total activity was 58% and 20% at substrate concentrations of 25 μM and 1500 μM, respectively. Zidovudine (a selective substrate for UGT2B7; Court et al., 2003) and fluconazole (a selective inhibitor of UGT2B7; Uchaipichat et al., 2006b) inhibited human liver microsomal LTG N2-glucuronidation by approximately 70% at the lower substrate concentration, and approximately 20% at the higher concentration. Nevertheless, a role for UGT1A1, 1A3, and 1A6 (the other hepatically expressed enzymes shown here to form LTG-Gluc) in the Hill component of human liver microsomal LTG N2-glucuronidation cannot be discounted. At present, selective inhibitors of these enzymes are unavailable. It should be noted that the involvement of UGT2B7 in LTG N2-glucuronidation is consistent with recent studies showing that this enzyme has the capacity to N-glucuronidate a number of drugs (Staines et al., 2004; Zhang et al., 2004). Interestingly, Staines et al. (2004) also reported very low activity (V_max = 0.79 pmol/min · mg) for carbamazepine N-glucuronidation by recombinant UGT2B7 with near complete inhibition of the human liver microsomal reaction by morphine and some other UGT2B7 substrates. Although this group suggested that carbamazepine and LTG were not glucuronidated by the same enzyme on the basis of minor inhibition of carbamazepine N-glucuronidation by LTG, the concentration of LTG used in the inhibition experiments (1000 μM) was low compared with the S₅₀ for the putative UGT2B7 component of LTG N2-glucuronidation reported here.

A recent study conducted in this laboratory (Uchaipichat et al., 2006b) demonstrated that BSA (2%) reduced the K_m for zidovudine (a selective UGT2B7 substrate) glucuronidation by HLMs and recombinant UGT2B7 approximately 10-fold, without affecting V_max. Here, BSA (2%) reduced the S₅₀ for the Hill component (proposed to be UGT2B7) of microsomal LTG N2-glucuronidation approximately 8-fold (without a significant effect on V_max or the Hill coefficient), but did not alter the derived kinetic constants for the Michaelis-Menten component of the microsomal reaction. In contrast, addition of BSA to incubations increased both the K_m and V_max for LTG N2-glucuronidation by recombinant UGT1A4. The reason for the differing effects of BSA on human liver microsomal and recombinant UGT1A4 is currently unknown. However, Tang et al. (2002) proposed that BSA reduces the K_m values of certain CYP2C9 substrates as a result of altered protein conformation or the “mopping up” of endogenous inhibitors present in microsomal incubations. Similar mechanisms may account for the effect of BSA on UGT2B7. A number of fatty acids are potent inhibitors of UGT2B7, with K_i values as low as 0.15 μM (Tsoutsikos et al., 2004). Hence, the addition of exogenous albumin to microsomal incubations may bind inhibitory fatty acids, with a reduction in the “apparent” K_m of the alternate substrate. It has also been reported recently that human serum albumin (HSA) was selected in yeast two-hybrid screening with UGT1A1 as the “bait” (Ohta et al., 2005). Furthermore, HSA bound to His-tagged UGT1A1 immobilized on a Ni-NTA resin, and an interaction between these proteins on the endoplasmic reticulum was hypothesized. Whether albumin interacts with UGT2B7 is currently under investigation in this laboratory. It should be noted that BSA and HSA both reduce the K_m (and K_i) values of UGT2B7 substrates to a similar extent (A. Rowland, P. I. Mackenzie, and J.O. Miners, manuscript in preparation).

Coadministration of VPA is known to reduce LTG clearance in vivo in a dose-dependent manner. In this study, VPA inhibited the Hill component of microsomal LTG N2-glucuronidation, but not the Michaelis-Menten component. Consistent with the hypothesis that UGT1A4 represents the Michaelis-Menten component, VPA did not inhibit LTG N2-glucuronidation by recombinant UGT1A4. VPA is a known substrate of UGT2B7 (Jin et al., 1993; Ethell et al., 2003), and VPA has previously been shown to inhibit zidovudine glucuronidation by UGT2B7 (Trapnell et al., 1998; Ethell et al., 2003). For experiments performed in the presence of Hec, addition of BSA (2%) caused a 6-fold reduction in the K_i for VPA inhibition of human liver microsomal LTG N2-glucuronidation, with no effect on the mechanism of inhibition (i.e., competitive).The reduction in K_i was associated with a significant improvement in the prediction of the magnitude of the LTG-VPA interaction in vivo, as also observed by Uchaipichat et al. (2006b) when predicting the extent of the fluconazole-zidovudine interaction. Using the mean K_i value generated in the presence of BSA and various estimates of total VPA concentration in vivo, the predicted LTG AUC ratio in the presence and absence of VPA ranged from 1.86 ([I]_average) to 2.31 ([I]_inlet.max). Morris et al. (2000) reported that the LTG AUC ratio increased, on average, by 2.6-fold in patients receiving VPA, 1000 mg/day (presumed to be 500 mg b.i.d.). In contrast, use of unbound VPA concentrations in the expression for the AUC ratio did not predict an interaction.

The nonhyperbolic kinetics for LTG N2-glucuronidation by HLMs observed here contrasts with a previous report of Michaelis-Menten kinetics (K_m 2560 μM, V_max 0.65 nmol/min · mg) for this pathway (Magdalou et al., 1992). Substrate concentration ranges differed between the two studies; 12 concentrations in the range 10 to 3000 μM in the present study versus 5 concentrations in the range 500 to 8000 μM in the previous work. To obtain substrate (LTG) concentrations above 3000 μM, the earlier study required a high content of DMSO (5% v/v) in microsomal incubations, and it is possible that this may selectively alter the activities of the microsomal UGTs that contribute to LTG N2-glucuronidation (Uchaipichat et al., 2004). Furthermore, transformation of the velocity versus substrate concentration data published by Magdalou et al. (1992) provides nonlinear Eadie-Hofstee plots inconsistent with Michaelis-Menten kinetics, suggesting that the original kinetic analysis may not have been appropriate.

In summary, both UGT1A4 and UGT2B7 appear to contribute to LTG N2-glucuronidation by human liver microsomes, with the UGT2B7-catalyzed reaction apparently dominating at low substrate concentrations. Given that the therapeutic plasma concentration range for LTG is 3 to 14 mg/l (12-55 μM), inhibition of UGT2B7-catalyzed LTG N2-glucuronidation provides a mechanism for the LTG-VPA interaction in vivo. VPA inhibits the UGT2B7, but not the UGT1A4, component of LTG N2-glucuronidation. Consistent with a recent report from this laboratory demonstrating that BSA reduced the K_m for zidovudine glucuronidation by human liver microsomal UGT2B7, addition of BSA (2%) to incubations caused a 6-fold reduction in the S₅₀ for the putative UGT2B7 component of LTG N2-glucuronidation. Similarly, significant inhibition of LTG N2-glucuronidation by VPA in vivo was predicted from the K_i value generated in the presence, but not the absence, of BSA. Together with the known effect of VPA on zidovudine glucuronidation both in vitro and in vivo (Lertora et al., 1994; Trapnell et al., 1998; Ethell et al., 2003), these data suggest that VPA may inhibit the metabolism of other substrates metabolized by UGT2B7, but not the glucuronidation of predominantly UGT1A4 substrates.