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CYP3A INDUCTION BY N-HYDROXYFORMAMIDE TUMOR NECROSIS FACTOR- CONVERTING ENZYME/MATRIX METALLOPROTEINASE INHIBITORS: USE OF A PREGNANE X RECEPTOR ACTIVATION ASSAY AND PRIMARY HEPATOCYTE CULTURE FOR ASSESSING INDUCTION POTENTIAL IN HUMANS

GlaxoSmithKline, Research Triangle Park, North Carolina (T.K.T., L.M., E.J.B., T.A.B., R.C.A., J.D.B., D.L.M., M.D.G., D.J.H., K.M.-K., J.L., S.K., S.N., R.L., Z.Z.); and Division of Drug Delivery and Disposition, University of North Carolina School of Pharmacy, Chapel Hill, North Carolina (T.K.T., G.H., S.J., E.L.L.)

(Received November 13, 2002; Accepted March 24, 2003)

	Abstract

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A series of N-hydroxyformamide tumor necrosis factor- converting enzyme (TACE)/matrix metalloprotease (MMP) inhibitors were evaluated for their potential to induce human cytochrome P450 3A (CYP3A). Two in vitro assays were used: 1) a cell-based reporter gene assay for activation of the pregnane X receptor (PXR), and 2) a primary "sandwich" culture of human hepatocytes. Approximately 50 TACE/MMP inhibitors were evaluated in the human PXR assay. A range of PXR activation was observed, 0 to 150% of the activation of the known human CYP3A inducer rifampicin. Three TACE/MMP inhibitors were evaluated in rat and human hepatocytes. Significantly higher PXR activation/CYP3A induction was observed in PXR/hepatocyte models, respectively, for (2R,3S) 3-(formyl-hydroxyamino)-2-(2-methyl-1-propyl)-4-methylpentanoic acid [(1S,2S)-2-methyl-1-(2-pyridylcarbamoyl)-1-butyl]amide (GW3333) compared with (2R,3S)-6,6,6-trifluoro-3-[formyl(hydroxy)amino]-2-isobutyl-N-{(1S,2R)-2-methoxy-1-[(1,3-thiazol-2-ylamino)carbonyl]propyl}hexanamide (GW6495) and (2R)-N-{(1S)-2,2-dimethyl-1-[(methylamino)carbonyl]-propyl}-2-{(1S)-1-[formyl(hydroxy)amino]ethyl}-5-phenylpentanamide (GI4023). The CYP3A induction level achieved with GW3333 at a concentration of approximately 10 µM in human hepatocytes was comparable to that achieved with rifampicin at a concentration of 10 µM. The extent of rodent CYP3A induction caused by GW3333 was confirmed in vivo after daily oral administration for 14 days to rats. In conclusion, GW3333 is a potential inducer of CYP3A expression in vivo in humans, but other N-hydroxyformamides are less likely to induce CYP3A.

An increasingly important aspect of drug safety evaluation is the determination of the potential for drug-drug interactions. This is especially important with new arthritic medications since the target population is likely to be under multiple drug therapy (Kovarik et al., 1997). Interactions involving CYP3A4 are particularly important to avoid because this enzyme plays an important role in the elimination of many marketed drugs (Maurel, 1996a; Thummel and Wilkinson, 1998). Drugs that inhibit or induce the levels of liver drug-metabolizing enzymes can alter the pharmacological and/or toxicological effects of the same agent and/or other drugs (Parkinson, 2001). For example, clinically important consequences of P450 enzyme induction include the enhanced biotransformation of cyclosporin and contraceptive steroids by inducers of the CYP3A enzymes, such as rifampicin and troglitazone (Sahi et al., 2000; Parkinson, 2001).

Historically, CYP3A enzyme induction has been evaluated by repeat daily dosing of the drug to rodents for several days. Animals are then euthanized and liver microsomes prepared from the treated animals. CYP3A activity is then quantitated by incubation of microsomes with known substrates of the CYP3A enzyme. However, drugs that induce rodent CYP3A do not always induce human CYP3A. For example, pregnenalone 16-carbonitrile (PCN) induces rat CYP3A but does not significantly induce human CYP3A (Kocarek et al., 1995; Jones et al., 2000). Conversely, rifampicin is a potent inducer of human CYP3A, but it does not induce rat CYP3A (Kocarek et al., 1995; Jones et al., 2000).

Recently, however, refinements in the isolation and culturing of hepatocytes (Maurel, 1996b; Hamilton et al., 2001), and the discovery that the CYP3A enzyme is regulated by the nuclear receptor, pregnane-X receptor (PXR) (Kliewer et al., 1998; Lehmann et al., 1998), have resulted in the availability of two useful in vitro assays to determine the potential of a compound to induce human CYP3A. Primary cultures of human hepatocytes, cultured in the sandwich configuration and treated with known inducers for 3 to 5 days, have been shown to induce CYP3A4 to an extent similar to that seen in vivo (Maurel, 1996b; LeCluyse et al., 2000). More recently, the PXR receptor has been found to be activated by the same compounds that induce CYP3A. For example, rifampicin activates human PXR but not rat PXR (Jones et al., 2000). Thus, CYP3A induction potential can now be determined in vitro in human-derived tissue and/or receptor assays.

The purpose of these studies was to determine the human CYP3A induction potential of the N-hydroxyformamide class of TACE/MMP inhibitors at the preclinical phase of the drug development process. By using the human-derived in vitro assays, PXR, and sandwich-cultured hepatocytes, we attempted to obtain an accurate rank order of the potential of the N-hydroxyformamide compounds to induce human CYP3A. Furthermore, by comparing the in vitro hepatocyte CYP3A induction caused by the most potent N-hydroxyformamide CYP3A inducer, GW3333, to the CYP3A induction caused by GW3333 in rats after in vivo treatment, we reasoned that we could assess whether therapeutic plasma concentrations of GW3333 in humans would cause CYP3A induction.

	Materials and Methods

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Chemicals and Reagents. GW3333 and other N-hydroxyformamide analogs were synthesized by the Medicinal Chemistry Department, GlaxoSmithKline. Chemical identity was confirmed by ¹H NMR, mass spectrometry, and elemental analysis. Chemical purity was determined by HPLC-MS to be greater than 95% for all compounds. All culture media and bovine insulin were obtained from Invitrogen (Carlsbad, CA). Matrigel, rat-tail collagen type I, and ITS+ culture supplement (625 µg/ml insulin, 625 µg/ml transferrin, 625 ng/ml selenium, and 100 mg/ml albumin) were from Collaborative Research (Bedford, MA). Petri dishes (60 mm) (Permanox) were from Nalge Nunc (Naperville, IL). Collagenase (type IV) and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO), except as noted below.

Cotransfection Assays. HuH7 cells were plated in 96-well plates at a density of 20,000 cells/well in phenol red-free DMEM/F-12 medium supplemented with 2 mM glutamine and 10% charcoal/dextran-treated fetal bovine serum (HyClone Laboratories, Logan, UT). Transfection mixes contained 5 ng of receptor expression vector (pSG5 hPXRATG or pSG5 rPXR) (Lehmann et al., 1998: Jones et al., 2000), 12 ng of reporter plasmid [CYP3A1(2X)tkSPAP containing two copies of the CYP3A1 PXR binding site upstream of the thymidine kinase minimal promoter and secreted placental alkaline phosphatase reporter] (Kliewer et al., 1998), 25 ng of pCH110 (ß-galactosidase) as internal control, and 23 ng of carrier plasmid. Transfections were performed with LipofectAMINE Plus (Invitrogen), essentially according to the manufacturer's directions, using 0.5 µl of LipofectAMINE and 0.7 µl of LipofectAMINE Plus reagent/well. Cells were transfected for 3 h in a volume of 100 µl. The transfection was terminated by adding 100 µl of phenol red-free DMEM/F-12 medium with 15 mM HEPES and supplemented with 20% charcoal-stripped, delipidated, heat-inactivated (62°C for 35 min) bovine calf serum (Sigma-Aldrich) and 4 mM glutamine to each well. Cells were returned to the incubator and allowed to recover overnight. Drug dilutions were prepared in phenol red-free DMEM/F-12 with 15 mM HEPES supplemented with 10% charcoal-stripped, delipidated, heat-inactivated bovine calf serum and 2 mM glutamine. Cells were incubated for 24 h in the presence of compounds, and then the medium was sampled and assayed for alkaline phosphatase activity. The remaining cells were lysed in the presence of 0.1% Triton X-100, and ß-galactosidase activity was measured. Reporter gene activity (secreted placental alkaline phosphatase) was normalized to the internal control (ß-galactosidase), and activities were plotted as percentage of maximum induction by 10 µM rifampicin (human) or 10 µM PCN (rat).

Isolation of Hepatocytes. All tissues were obtained through qualified medical staff, with donor consent and with the approval of the University of North Carolina Hospitals ethics committee. Hepatocytes were isolated from human liver tissue procured through the Department of Surgery, University of North Carolina at Chapel Hill School of Medicine, or from nontransplantable donor livers by a modification of the two-step collagenase digestion method of MacDonald et al. (2001). Prior to perfusion of the tissue, the outer capsule on the cut surface was reconstructed utilizing medical grade adhesive. This technique permitted the use of lower flow rates and enhanced both blood clearance and digestion of the liver. In most cases, encapsulated liver tissue (15–100 g) was perfused with calcium-free buffer containing 5.5 mM glucose, 0.5 mM EGTA, 50 mg/ml ascorbic acid, and 0.5% bovine serum albumin for 10 to 15 min at a flow rate of 15 to 30 ml/min, followed by Dulbecco's modified Eagle's medium (DMEM) containing 0.5% bovine serum albumin, ascorbic acid (50 mg/ml), and collagenase (0.4–0.8 mg/ml) for 15 to 20 min at a flow rate of 15 to 30 ml/min.

Hepatocytes were dispersed from the digested liver in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum, insulin (4 µg/ml), and dexamethasone (1.0 µM), passed through a series of fluorocarbon filters (1000-, 400-, and 100-µm mesh), and washed by low-speed centrifugation (70g, 4 min). Cell pellets were resuspended in 30 ml of supplemented DMEM and 8 to 12 ml of 90% isotonic Percoll, and centrifuged at 100g for 5 min. The resulting pellets were resuspended in fresh medium and washed once by low-speed centrifugation. Hepatocytes were resuspended in supplemented DMEM, and viability was determined by trypan blue exclusion. Cells were not used for experiments unless the viability was at least 75%.

Primary Cultures of Hepatocytes. Both rat and human hepatocytes were cultured according to the methods described by LeCluyse et al. (2000). In most cases, 4 x 10⁶ hepatocytes were added to 60-mm culture dishes precoated with type I collagen in 3 ml of supplemented DMEM and allowed to attach for 3 to 6 h at 37°C in a humidified incubator with 95% air/5% CO₂. After cell attachment, culture dishes were gently swirled, and medium containing unattached cells was aspirated and replaced with modified Chee's medium containing ITS+ culture supplement (1:100) and 0.1 µM dexamethasone (LeCluyse et al., 1999, 2000). Medium was changed on a daily basis thereafter.

In Vitro Enzyme Induction in Rat and Human Hepatocytes. Primary cultures of hepatocytes were maintained for 36 to 48 h before initiating treatment with test compounds. Cultures were treated for 3 consecutive days with prototypical inducers or TACE/MMP inhibitors over a range of concentrations (0.1–250 µM). Unless otherwise specified, control cultures were treated with cell culture media that contained 0.1% DMSO.

Cell Harvest and Microsome Preparation. At the end of 3 days of treatment, cultures from each experimental group were rinsed twice with ice-cold Hanks' buffered saline solution. Homogenization buffer (50 mM Tris-HCl, pH 7.0, 150 mM KCl, 2 mM EDTA) was added to each dish (total of 2–3 ml per group), and cells were scraped with a rubber policeman. Harvested cells from each group were then pooled and sonicated with a Vibra-Cell probe sonicator (Sonics & Materials Inc., Newton, CT) at 40 W for 15 to 20 s (Wortelboer et al., 1990). Cell lysates were centrifuged at 9,000g for 20 min at 4°C. Supernatants were collected and centrifuged at 100,000g for 60 min at 4°C. The final microsomal pellets were resuspended in 100 to 300 µl of 0.25 M sucrose with the aid of a Potter-Elvehjem tissue grinder fitted with a Teflon pestle. Duplicate aliquots (5 µl) were removed for protein determination before samples were stored at –80°C.

Protein Determination. The protein content in each sample was determined with a commercially available kit (BCA Protein Assay, Pierce Chemicals, Rockford, IL), based on instructions provided by the manufacturer. Bovine serum albumin served as standard (supplied with the BCA kit).

Determination of CYP3A Activity in Microsomes. CYP3A activity was assessed by incubating microsomal samples with the cofactor NADPH at a concentration of 1 mM and the substrate testosterone at a concentration of 250 µM (Pearce et al., 1996). After a 12-min incubation period, the reaction was quenched by adding 0.5 volume of acetonitrile. The resulting extracts were analyzed using HPLC with UV detection at 240 nm. Testosterone and its metabolites were resolved using a Waters Symmetry C18 column, 3 x 150 mm, using a mobile phase consisting of mixtures of aqueous solvent containing 0.1% formic acid, 0.25% triethylamine, and acetonitrile. The percentage of acetonitrile was changed from 25 to 40% over a 4 min linear gradient and then held isocratic for an additional 4 min. The retention time of 6ß-hydroxy testosterone was 3.8 min and was separated from the nearest testosterone metabolite, 16-hydroxytestosterone, by approximately 0.5 min. HPLC-UV responses were used to construct a peak area ratio (6ß-hydroxy testosterone peak area/internal standard peak area) versus concentration calibration curve. CYP3A activity is reported as picomoles of 6ß-hydroxy testosterone per minute per milligram of microsomal protein. Each microsomal sample was assayed in duplicate.

Animal Dosing and Sample Collection. Male GLX Wistar Han rats were purchased from Charles River (Raleigh, NC) and were acclimated for at least 1 week before dosing. GW3333 was suspended in an aqueous vehicle that contained 0.5% hydroxypropylmethylcellulose and 0.1% Tween 80. The final dosing volume was 10 ml/kg body weight. Animals were dosed once daily for 14 days with GW3333 at dose levels of 50, 150, or 500 mg/kg body weight. Each dose group contained six animals. Control animals received hydroxypropylmethylcellulose/Tween 80 vehicle.

Blood samples were collected for GW3333 concentration measurement from six rats on the first and last day of dosing via one of the lateral tail veins at 0.5, 1.5, 4, 8, and 24 h after dose administration. Each animal was sampled at only two of the time points listed above. Blood was centrifuged to obtain plasma.

Twenty-four hours after the last dose on day14, the animals were euthanized, and the livers were removed and weighed. Slices of liver tissue were fixed in 2.5% glutaraldehyde in sodium cacodylate buffer and examined using transmission electron microscopy. Additional liver slices were fixed in 10% neutral buffered formalin, sectioned, and stained with H&E and examined by light microscopy. The remainder of the liver tissue was stored frozen until later processing to obtain microsomes as described above for hepatocytes. CYP3A activity was measured in microsomal samples from each animal as described above. Mean CYP3A activity was determined for each treatment group.

Determination of GW3333 Plasma Concentration. Plasma samples were prepared for analysis by extraction with 4 volumes of acetonitrile that contained an internal standard. After centrifugation, the supernatant was evaporated to dryness and then dissolved in HPLC mobile phase. GW3333 was eluted from an HPLC column (Hypersil C18, 4.6 x 100 mm, 3 µm) using a mobile phase consisting of 10 mM ammonium acetate (pH 4.5) and acetonitrile. GW3333 product ion (M + H = 421 m/z) was measured using a Finnigan 700 MS after ionization by atmospheric pressure chemical ionization. Plasma standards were prepared by adding GW3333 to blank plasma. HPLC-MS responses were used to construct a peak area ratio (GW3333 peak area/internal standard peak area) versus concentration calibration curve. A composite GW3333 plasma concentration versus time curve was constructed from all animals within a treatment group. The composite area under the plasma concentration-time curve (AUC) was calculated by the trapezoidal method.

Statistical Analyses. The statistical testing of in vitro PXR activation and CYP3A induction was facilitated by fitting a smooth spline curve to model the dose-response relationship for each N-hydroxyformamide compound using "gam" function with the "tp" option from library mgcv in R version 1.6.2 (Ihaka and Gentleman, 1996). To detect whether the activity of any concentration for a single drug compound differed from that of the nondrug-treated cells, the spline model fit was compared with the model fit assumed under no drug activity (a horizontal line with no fold increase over the negative control) via a lack-of-fit F test. To assess the minimum drug concentration that caused a significant elevation in the enzyme activity, 95% confidence limits were computed for each concentration. If the confidence limits for the enzyme activity at a particular concentration did not include the activity of the nondrug-treated cells, then that concentration was considered to be significantly different from the negative control. In addition, where appropriate, EC₅₀ values were calculated using the spline model. Comparisons of any two compounds at chosen concentrations were also made via the spline models. The mean response and associated standard error were obtained from each spline model so that comparisons could be made via a Welch two-sample t test.

Where possible, comparisons were also made via analysis of variance (ANOVA) modeling. For the PXR activation data, comparisons were made with both ANOVA and spline modeling. Conclusions drawn from ANOVA matched those from spline fits and were, therefore, not reported. For in vivo studies, differences between mean CYP3A activities were compared using ANOVA modeling.

	Results

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In Vitro Human and Rat PXR Activation. Activation of PXR by N-hydroxyformamide compounds was determined in a cell-based transient transfection assay. A wide range of human PXR activation was observed for the TACE/MMP inhibitors at a concentration of 10 µM (Fig. 1). The compound that caused the highest level of human PXR activation in these experiments was GW3333 (Fig. 1). Three compounds with different efficacies toward hPXR activation were selected for more detailed transfection analysis (Fig. 2). All three TACE/MMP inhibitors caused a significant activation of hPXR (Fig. 3) at concentrations between 0.2 and 0.5 µM. However, at the maximum testable concentration of 10 µM GW3333, GI4023 and GW6495 caused activation to a different extent, achieving activation of 80%, 40%, and 30%, respectively, of that caused by 10 µM rifampicin. At this concentration, GW3333 caused a significantly higher PXR activation compared with GW6495 and GI4023. For the known hPXR activator, rifampicin, the hPXR activation was significantly higher than that of control treated cells at a concentration of approximately 0.1 µM, and at the highest tested concentration of 10 µM, achieved significantly higher hPXR activation relative to GW3333. The hPXR activation caused by the TACE/MMP inhibitors did not appear to reach a plateau at a concentration of 10 µM; thus, an accurate EC₅₀ was not calculable for the N-hydroxyformamide analogs. The EC₅₀ value for rifampicin was 1 µM for human PXR activation.

View larger version (41K): [in this window] [in a new window]   FIG. 1. Human PXR activation by 49 N-hydroxyformamide TACE/MMP inhibitors. Activation of human PXR by N-hydroxyformamide compounds was determined at a single concentration of 10 µM in a cell-based transient transfection assay as described under Materials and Methods. The extent of PXR activation by each N-hydroxyformamide compound is reported as a percentage of the hPXR activation observed for the known hPXR activator rifampicin at a concentration of 10 µM.

View larger version (9K): [in this window] [in a new window]   FIG. 2. Structures of selected N-hydroxyformamide TACE/MMP inhibitors.

View larger version (14K): [in this window] [in a new window]   FIG. 3. Human and rat PXR activation by GW3333, GW6495, and GI4023. Activation of human and rat PXR by N-hydroxyformamide compounds was determined at multiple concentrations in a cell-based transient transfection assay as described under Materials and Methods. The extent of human or rat PXR activation by each N-hydroxyformamide compound is plotted as a percentage of the PXR activation observed for the known hPXR activator rifampicin or the known rPXR activator PCN, respectively, at a concentration of 10 µM. A smooth spline curve was fit to the data for each TACE/MMP inhibitor, and a significant elevation of PXR activity was determined as described under Materials and Methods. All TACE/MMP inhibitors significantly activated rat PXR at concentrations of 0.2 to 0.6 µM, and human PXR at concentrations of 0.2 to 0.5 µM. The known PXR activators PCN and rifampicin significantly activated rat and human PXR, respectively, at concentrations of 0.02 µM and 0.08 µM. Asterisks indicate a significantly different (p < 0.001) PXR activation by GW3333 relative to GI4023 and GW6495 as well as the positive PXR activators rifampicin and PCN at a concentration of 10 µM using a Welch two-sample t test.

As observed in cells transfected with hPXR, all three TACE/MMP inhibitors caused a significant activation of rPXR at concentrations between 0.2 and 0.6 µM as assessed by spline analysis. At 10 µM, GW3333 caused significantly higher rPXR activation compared with the other two TACE/MMP inhibitors (Fig. 3), activating rPXR to approximately 50% of the activation caused by PCN at a concentration of 10 µM. For the known rPXR activator PCN, the rPXR activation was significantly higher than control treated cells at a concentration of approximately 0.02 µM, and at the highest tested concentration of 10 µM, achieved significantly higher rPXR activation relative to GW3333. The EC₅₀ value for PCN was 0.4 µM for rat PXR activation.

In Vitro CYP3A Induction in Rat and Human Hepatocytes. Induction of CYP3A by the selected N-hydroxyformamide TACE/MMP inhibitors shown in Fig. 2 was determined after daily treatment of human and/or rat sandwich-cultured hepatocytes over a concentration range of 0.1 to 250 µM for 3 days. CYP3A activity was measured in microsomes prepared from each treatment group. Basal 6ß-hydroxy testosterone formation rates in control human hepatocytes and the magnitude of increase in the presence of rifampicin varied considerably (Table 1). Despite this variability, which is thought to reflect the normal variability present in the human population (Maurel, 1996b; LeCluyse, 2001), a clear increase in CYP3A activity was observed in all TACE/MMP inhibitor-treated human and rat hepatocytes (Tables 1 and 2). In human hepatocytes, GW3333, at concentrations of approximately 10 µM, induced increases in CYP3A activity approximately comparable to that seen with 10 µM rifampicin. In rat hepatocytes, GW3333 caused increases in CYP3A activity of approximately 7-fold relative to control hepatocytes (Table 2). This CYP3A activity was even higher than that caused by the known rodent CYP3A inducer PCN at a concentration of 1 µM. However, the relatively modest 2-fold induction by 1 µM PCN is likely due to insufficient PCN concentrations. In subsequent experiments with different hepatocytes, treatment with 10 µM PCN resulted in increased CYP3A activity of 10- to 13-fold relative to control hepatocytes (data not shown).

Since concentrations up to 250 µM TACE/MMP inhibitors were tolerated by the sandwich-cultured hepatocytes, a maximum induction response for the TACE/MMP inhibitors appeared achievable in the hepatocyte assay. For GW3333, the maximum CYP3A activity in both rat and human hepatocytes occurred at a concentration of approximately 10 µM. The EC₅₀ for CYP3A induction by GW3333 was estimated to be 1.8 µM and 4.0 µM in human and rat hepatocytes, respectively. Two other N-hydroxyformamide analogs, GW6495 and GI4023, also caused a significant increase in CYP3A activity in rat and human hepatocytes (Tables 1 and 2). However, these compounds did not achieve CYP3A induction levels comparable to those of GW3333 in human or rat hepatocytes even at the maximum observed induction levels for these compounds, a concentration as high as 100 µM for GI4023 and 25 µM for GW6495.

In Vivo Enzyme Induction in Rats. After daily oral administration of GW3333 to rats for 14 days at doses of 50, 150, and 500 mg/kg, no changes in body weight were observed. However, liver weights of treated animals did increase in a dose-related manner (Table 3). Light microscopic examination of liver tissue revealed dose-related increases in vacuolation and hepatocyte hypertrophy at the 150 and 500 mg/kg dose levels (data not shown). Electron microscopic examination revealed that the hypertrophy was primarily due to proliferation of smooth endoplasmic reticulum and that there was also an increase in intracellular lipids in the 150 and 500 mg/kg dose groups.

GW3333 plasma AUC values determined on day 14 were reduced by 40, 50, and 61% for animals treated with 50, 150, and 500 mg/kg, respectively, compared with those determined on day 1. Likewise, C_max values were decreased by 17, 42, and 55% at day 14 in the respective animals (Table 3).

Determination of the CYP3A enzyme activity in microsomes that were prepared from GW3333-treated and control animal liver tissue showed a significant increase (p < 0.001) in CYP3A activity as measured by an increase in formation rate of 6ß-hydroxy testosterone in all GW333 treatment groups compared with the control group (Fig. 4). A significant dose-dependent increase in CYP3A activity was observed in animals treated with GW3333, when comparing the 500 mg/kg treatment group to the 50 mg/kg treatment group (p < 0.05).

View larger version (53K): [in this window] [in a new window]   FIG. 4. Increase in CYP3A activity in microsomes from rats dosed once daily for 14 days with GW3333. Microsomes were prepared from livers collected 24 h after the last daily dose of vehicle or GW3333. Production of the CYP3A-specific metabolite, 6ß-hydroxy testosterone, was measured by LC/MS-MS after incubation of microsomes with testosterone as described under Materials and Methods. CYP3A activity is reported as the rate of 6ß-hydroxy testosterone formed per milligram of protein. The numbers on the bars indicate the fold induction of CYP3A activity above that observed in microsomes from control rats. Asterisks indicate a significant (p < 0.001) difference from vehicle treatment, and the number sign (#) on the 500 mg/kg dose group indicates a significant difference (p < 0.05) from the 50 mg/kg treatment group based on ANOVA modeling.

	Discussion

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The studies reported herein clearly indicate that N-hydroxyformamide compounds, in particular GW3333, cause CYP3A induction in vitro in rat and human cells. In vitro incubation of N-hydroxyformamide compounds with transfected cells containing rat and human PXR resulted in varied increases in activation of PXR (Figs. 1 and 3). The range of activation observed with the 49 TACE/MMP inhibitors in the human PXR activation assay (Fig. 1) suggests that PXR activation is not a characteristic for all molecules in this structural class. The most potent N-hydroxyformamide, GW3333, caused a strong activation of hPXR at 10 µM. However, the onset of PXR activation by rifampicin began at a slightly lower concentration (0.1 µM) and caused a significantly higher activation at 10 µM. Thus, rifampicin is a more potent activator of hPXR compared with GW3333 and the other TACE/MMP inhibitors. Likewise, PCN caused activation of rPXR at a lower concentration (0.02 µM) and caused a significantly higher activation at 10 µM compared with GW3333. Thus, PCN is a more potent activator of rPXR compared with GW3333 and the other TACE/MMP inhibitors.

To further assess the PXR activation / CYP3A induction caused by the N-hydroxyformamide compounds, a second in vitro assay, primary cultures of hepatocytes, was carried out. N-hydroxyformamide compounds were selected based on their differences in PXR activation potency, as well as their ability to inhibit the target TACE/MMP enzymes. Each compound was incubated in rat and human sandwich-cultured hepatocytes for 3-days. As seen in the PXR assay, GW3333 caused a significantly higher CYP3A induction at 10 µM (Tables 1 and 2) compared with the other two TACE/MMP inhibitors. Thus, both assays identified GW3333 to have the highest PXR activation/CYP3A induction activity in the series of three TACE/MMP inhibitors that were tested in both assays. The identification of GW3333 as the most potent N-hydroxyformamide to activate PXR and to induce CYP3A in sandwich-cultured hepatocytes is consistent with the large amount of data in the literature that supports PXR regulation of CYP3A (Bertilsson et al., 1998; Blumberg et al., 1998; Kliewer et al., 1998; Lehmann et al., 1998; Jones et al., 2000).

Upon closer inspection of the data obtained from the PXR and hepatocyte assays, some quantitative differences can be observed in the extent of PXR activation and CYP3A induction versus the known CYP3A inducer rifampicin. For example, GW3333 did not achieve the same PXR activation as rifampicin at a concentration of 10 µM (p < 0.05), whereas in the hepatocyte assay, GW3333 appeared to achieve fold induction of CYP3A comparable to that of rifampicin at approximately 10 µM (no statistical test performed). Lower PXR activation for GW6495 was also observed compared with the extent of CYP3A induction by rifampicin in hepatocytes. Therefore, although both assays identified GW3333 as the most potent CYP3A inducer in this series of three TACE/MMP inhibitors, the extent of CYP3A induction versus rifampicin differed between the two assays.

Interestingly, since CYP3A induction was observed in both rat and human hepatocytes, particularly with GW3333, it appears that the N-hydroxyformamide compounds are not species-specific CYP3A inducers like PCN or rifampicin. Therefore, GW3333 might be a useful probe to induce CYP3A in rat, human, and possibly other species.

We sought to further assess the in vitro CYP3A induction response of GW3333 in an animal model. This compound was chosen based on its in vitro CYP3A induction potency, as well as its favorable in vivo pharmacokinetic properties (data not shown). Furthermore, because GW3333 had shown efficacy in rat models of arthritis (Conway et al., 2001), the measurement of in vivo induction with GW3333 in the same animal would allow us to relate efficacious concentrations to those that induce CYP3A. Clearly, repeat daily oral administration of GW3333 to rats at doses of 50, 150, and 500 mg/kg (10-, 30- and 100-fold higher than the efficacious doses of GW3333) did result in several dose-related changes that suggested liver enzyme induction. At all doses, a dose-dependent increase in liver weight and a decline in drug plasma concentrations were observed (Table 3). Proliferation of smooth endoplasmic reticulum, the cellular location of the P450 enzymes, was observed in the two highest treatment groups.

At least one P450 isoform, CYP3A, was induced by daily oral dosing with GW3333 to rats as shown by the increase in CYP3A-mediated formation of 6ß-hydroxy testosterone in microsomal incubations (Fig. 4). There was a slight increase in CYP3A activity over the dose range 50 mg/kg to 500 mg/kg. The CYP3A induction by GW3333 of 3-fold compared with nontreated animals is similar to that reported for the prototypical rodent CYP3A inducer, PCN, after oral dosing to rats (Sonderfan et al., 1987). In addition to CYP3A, CYP2B was also induced by daily treatment with GW3333, because increased formation of CYP2B-specific metabolite 16ß-hydroxytestosterone (Sonderfan et al., 1987) was observed also in microsomes from GW3333-treated animals (data not shown).

The metabolic pathway(s) for GW3333 have not been fully elucidated; however, evidence for the metabolism and elimination of GW3333 by P450 enzymes was obtained in separate experiments using liver microsomes obtained from nontreated rats. In these studies, GW3333 was degraded by rat liver microsomes in the presence of P450 cofactor NADPH but was not significantly degraded by microsomes that did not contain NADPH (data not shown). Recombinant human CYP3A also caused appreciable in vitro degradation of GW3333 (data not shown). Thus, these data support the conclusion that the decrease in GW3333 plasma concentrations after repeated daily treatment with GW3333 to rats is due at least in part to induction of CYP3A, which in turn leads to an increase in its own metabolism and elimination. The metabolic pathway(s) of GW3333 are currently under investigation in our laboratory.

On the basis of these in vitro and in vivo studies, we have clearly established that GW3333 is a potent inducer of rat and human CYP3A. However, we were interested in estimating the in vivo human response to this compound prior to its clinical testing. As a basis for these approximations, we compared the rat in vitro hepatocyte CYP3A induction to the in vivo CYP3A induction response, and also compared the corresponding GW3333 concentrations in each model system. The GW3333 plasma concentration maximum (C_max) after daily-repeat oral dosing to rats ranged between 10 and 50 µM. These concentrations of GW3333 caused a CYP3A induction of approximately 3-fold relative to control animals. In rat hepatocytes, these concentrations of GW3333 caused a CYP3A induction of approximately 6-fold and a significant induction of CYP3A was observed at an even lower GW3333 concentration (1.2 µM). It is not known why rat hepatocytes appeared to achieve higher levels of CYP3A activity relative to animals that had similar GW3333 plasma concentrations. One plausible explanation would be that higher GW3333 concentrations were achieved in vitro in hepatocytes versus those achieved in vivo due to high plasma protein binding of GW3333. It can be concluded, however, that a significant CYP3A induction was observed both in vivo and in vitro at GW3333 concentrations of 10 µM.

As previously reported (Conway et al., 2001), GW3333 inhibited TACE and symptoms of inflammation after oral dosing to rats. TACE was inhibited at GW3333 plasma concentrations (C_max) as low as 0.5 µM. However, to suppress arthritic symptoms in chronic models of arthritis such as the rat peptidoglycan polysaccharide polymers model or the rat adjuvant arthritis model, plasma C_max values >10 µM were required (Conway et al., 2001). Given that a robust CYP3A induction was caused by GW3333 in vitro and in vivo in rats at this concentration, and that comparable CYP3A induction was caused by GW3333 in human hepatocytes at approximately the same concentrations, it is entirely likely that CYP3A induction would occur at therapeutic GW3333 plasma concentrations in humans.

In summary, we have presented the utility of the PXR activation assay and primary cultures of hepatocytes to evaluate the human CYP3A induction potential of N-hydroxyformamide compounds at an early stage in the drug development process. Furthermore, we have demonstrated that GW3333 is the most potent inducer in this series for CYP3A induction response. Other N-hydroxyformamide TACE/MMP inhibitors are likely to cause much lower CYP3A induction, based on the PXR activation and CYP3A induction results with these compounds.

	Acknowledgments

 
We gratefully acknowledge Mike McNulty for helpful discussions, Mike Emptage for statistical advice, and Charley Boehlert, Scott Barros, Rick Graham, and Patrick Finn for technical assistance.

	Footnotes

 
This work was financed by GlaxoSmithKline, Research Triangle Park, North Carolina.

¹ Abbreviations used are: TACE, tumor necrosis factor- converting enzyme; MMP, matrix metalloproteinase; GW3333, (2R,3S) 3-(formyl-hydroxyamino)-2-(2-methyl-1-propyl)-4-methylpentanoic acid [(1S,2S)-2-methyl-1-(2-pyridylcarbamoyl)-1-butyl]amide; PCN, pregnenalone16-carbonitrile; PXR, pregnane X receptor; HPLC, high-performance liquid chromatography; MS, mass spectrometry; DMSO, dimethyl sulfoxide; AUC, area under the plasma concentration-time curve; ANOVA, analysis of variance; h, human; GI4023, (2R)-N-{(1S)-2,2-dimethyl-1-[(methylamino)carbonyl]propyl}-2-{(1S)-1-[formyl(hydroxy)amino]ethyl}-5-phenylpentanamide; GW6495, (2R,3S)-6,6,6-trifluoro-3-[formyl(hydroxy)amino]-2-isobutyl-N-{(1S,2R)-2-methoxy-1-[(1,3-thiazol-2-ylamino)carbonyl]propyl} hexanamide; r, rat; P450, cytochrome P450; LC/MS-MS, liquid chromatography/tandem mass spectrometry.

Address correspondence to: Timothy K. Tippin, Drug Metabolism and Pharmacokinetics Department, Metabolic and Viral Diseases Center of Excellence in Drug Discovery, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709. E-mail Tim.K.Tippin{at}gsk.com

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