Introduction

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Tazarotene (Fig. 1), a novel acetylenic retinoid, is known to be effective in the topical treatment of psoriasis and acne (Weinstein, 1997; Shalita et al., 1999). Tazarotene, a prodrug, was designed to undergo rapid and complete metabolism to its active metabolite tazarotenic acid (Fig. 1). The exact mechanism of action of tazarotenic acid in the treatment of psoriasis and acne is unknown. However, it is thought that the selective interaction of tazarotenic acid with retinoic acid receptor (RAR¹), in particular RAR and RAR subtypes and the subsequent induction of both positive and negative gene regulatory effects may be involved (Nagpal et al., 1995; Chandraratna, 1996; DiSepio et al., 1998). Thus, tazarotenic acid modulates the pathogenic factors of psoriasis through normalizing abnormal keratinocyte differentiation and has potent anti-hyperproliferative effects in skin and decreases inflammation (Chandraratna, 1996).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Chemical structures of tazarotene, tazarotenic acid, the sulfoxide metabolite of tazarotenic acid, the sulfone metabolite of tazarotenic acid, and internal standard.

Following topical administration of tazarotene to healthy volunteers or patients with psoriasis or acne vulgaris, low but detectable plasma levels of tazarotenic acid can be measured (Tang-Liu et al., 1999). Oral tazarotene is currently in clinical development for the treatment of plaque psoriasis and acne. The rapid blood hydrolysis of tazarotene to the more water soluble tazarotenic acid is mediated by one or more paraoxon-inhibitable esterases (Madhu et al., 1997). In rat and human liver microsomes, the biotransformation of tazarotene was independent of cytochrome P450 (Tang-Liu et al., 1999). A recent pharmacokinetic study to assess the safety and dose proportionality of oral tazarotene found the main circulating agent, tazarotenic acid, was rapidly available in the systemic circulation with T_max ranging from 2- to 3-h postdose (Tang-Liu et al., 1999). Tazarotenic acid is subsequently oxidized to the inactive sulfoxide metabolite, the major metabolite excreted in urine (Tang-Liu et al., 1999).

The possibility for drug interactions occurs whenever two or more drugs are given concurrently. Since the metabolism of a drug usually represents a major route of elimination for that drug, it is important to be able to predict potential drug-drug interactions. A number of common in vitro approaches have been developed to identify which cytochrome P450 isozymes are responsible for metabolizing drugs in humans. These methods include the use of selective inhibitors with microsomes and the monitoring of catalytic activity in cDNA-based vector systems (Lin and Lu, 1997). The present in vitro study was conducted to identify the human microsomal enzyme(s) responsible for the metabolism of tazarotenic acid to its sulfoxide metabolite.

	Materials and Methods

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Chemicals and Reagents. Tazarotenic acid, the sulfoxide metabolite of tazarotenic acid, the sulfone metabolite of tazarotenic acid and internal standard 4-(4,4-dimethylchroman-6-ylethynyl)-benzoic acid (AGN 190252) were provided by Allergan, Inc. (Irvine, CA). The following chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO): paclitaxel, 9-cis-retinoic acid, 13-cis-retinoic acid, quercetin, potassium phosphate monobasic, potassium phosphate dibasic, -NADP, -NAD, and glucose 6-phosphate monosodium salt. Glucose-6-phosphate dehydrogenase was purchased from Roche Diagnostics (Indianapolis, IN). Ammonium acetate and magnesium chloride were purchased from Mallinckrodt Specialty Chemical Co. (St. Louis, MO). All other chemicals and reagents were of HPLC grade unless otherwise noted.

Human Microsomes. Pooled human liver microsomes from 15 donors were obtained from XenoTech LLC (Kansas City, KS) and phenotyped by the supplier for P450-specific activities. Microsomes prepared from human B-lymphoblastoid cell lines or baculovirus insect cell lines engineered to stably express specific human liver microsomal isoforms were obtained from BD Gentest Corporation (Woburn, MA).

Experimental Protocols.

Incubation method optimization The incubation conditions were first optimized in preliminary experiments using pooled human liver microsomes by varying the substrate concentration (1 to 200 µM), the incubation time (20 to 90 min) and the microsomal protein content (0.25 to 1 mg/ml). The optimized incubation conditions were chosen based on the formation of the sulfoxide metabolite from tazarotenic acid in human liver microsomes. The microsomal reaction mixture contained 10 µM tazarotenic acid, 1 mg/ml human liver microsomal protein, NADPH-regenerating system (0.4 mM NADP, 4 mM glucose 6-phosphate, 2 mM MgCl₂, 0.6 units/ml glucose-6-phosphate dehydrogenase and 1 mM NAD) and 0.1 M potassium phosphate buffer (pH 7.4). Incubations were conducted in final volume of 2 ml, in a water bath shaker (Precision, Chicago, IL), at 37°C, for 30 min. The reaction was terminated by transferring a 400-µl aliquot of microsomal reaction mixture to an Eppendorf tube containing 400 µl of ice-cold acetonitrile and 10 µl of 100 µg/ml internal standard. The resulting sample (0.8 ml) was vortexed and centrifuged at 1000g for 10 min to remove protein. The supernatant was evaporated to dryness under nitrogen; the dried residue was reconstituted in 0.4 ml of mobile phase (20% ACN, 80% 10 mM ammonium acetate/0.5% acetic acid, pH 3.4) and centrifuged at 1000g for 10 min. The supernatant (100 µl) was injected onto the HPLC system.

Metabolism of tazarotenic acid by cDNA-expressed P450 isozymes. Microsomes (1 mg/ml) prepared from human B-lymphoblastoid cell lines containing specific human cytochrome P-450 isozymes were incubated with 10 µM tazarotenic acid, for 30 min, at 37°C, in the presence of an NADPH-regenerating system, in a final volume of 1 ml. The microsomes contained CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP4A11. All incubations were performed in duplicate on two separate occasions.

Chemical inhibition of CYP2C8 in pooled human liver microsomes. Inhibition experiments using paclitaxel, 9-cis-retinoic acid, 13-cis-retinoic acid and quercetin as inhibitors of CYP2C8, were conducted in a similar manner. CYP2C8 is believed to mediate the metabolism of paclitaxel (Taxol), 9-cis-retinoic acid (Panretin), and 13-cis-retinoic acid (Accutane) (Parkinson, 1996; Mugford and Kedderis, 1998; Marill et al., 2002). Quercetin is a specific inhibitor of CYP2C8 (Parkinson, 1996). Both the inhibitor and tazarotenic acid were added to pooled human liver microsomes (1 mg/ml) before addition of cofactors in a total volume of 0.5 ml and incubated for 30 min, at 37°C.

Metabolism of tazarotenic acid by cDNA-expressed FMO enzymes. Microsomes (0.25 mg/ml) prepared from baculovirus insect cell lines containing human flavin-containing monooxygenase (FMO) isozyme were incubated with 10 µM tazarotenic acid, for 30 min, at 37°C, in the presence of an NADPH-regenerating system, in a final volume of 1 ml. The microsomes contained FMO1 (found in human fetal livers) or FMO3 (found in adult human livers) (Cashman, 1995). All incubations were performed in duplicate on two separate occasions.

Chemical inhibition of FMO in pooled human liver microsomes. Inhibition experiments to determine the IC₅₀ of methimazole as an inhibitor of FMO activity were conducted by adding both inhibitor and tazarotenic acid to 0.25 mg/ml pooled human liver microsomes and incubating for 90 min, at 37°C, in a total volume of 2 ml. The concentration of tazarotenic acid was 10 µM. Methimazole concentrations varied from 1 to 1000 µM. A control incubation containing no inhibitor was performed. All incubations were performed in duplicate on two separate occasions.

Bioanalysis.

HPLC analysis Authentic standards of the sulfoxide metabolite of tazarotenic acid, the sulfone metabolite of tazarotenic acid, tazarotenic acid, and internal standard were analyzed to establish the retention times of these compounds (Fig. 2). The sulfoxide metabolite could potentially be oxidized to its sulfone analog. However, sulfone metabolite formation was not observed in our incubation experiments (Fig. 2). The calibration curve for the metabolite concentration range of 0.025 to 20 µg/ml was linear with a correlation coefficient of 0.999. 

View larger version (13K): [in this window] [in a new window]   Fig. 2.   HPLC-UV chromatograms of the sulfoxide metabolite of tazarotenic acid, the sulfone metabolite of tazarotenic acid, and tazarotenic acid standards in initial mobile phase (top panel) and an incubation sample in which 10 µM tazarotenic acid was incubated for 30 min, at 37°C, with 1 mg/ml pooled human liver microsomes (bottom panel).

Mass spectral analysis. Mass spectral analyses were performed on a PE-Sciex API 365 tandem mass spectrometer (Applied Biosystems, Foster City, CA). The liquid chromatographic separation was performed on a reversed-phase HPLC column (LUNA C-18, 2.0 mm x 30 mm, 3 µm particle size; Phenomenex Torrance, CA) eluted at 0.2 ml/min using a similar HPLC gradient system. The mass spectrometer was operated in positive ion mode of electrospray method with full scan from m/z 67 to 350.

Data Analysis. GraFit Version 4.0 (Erithacus Software Limited, London, UK) was used for determining K_m, V_max, IC₅₀ and K_i values. Depending on the study type, each data set obtained from an incubation experiment was fitted to the appropriate enzyme kinetics equation by nonlinear regression analysis. Each data set included mean values collected from two separate determinations performed in duplicate each time (i.e., for a total of four replicates). To ensure these parameters were determined during linear enzyme kinetics, assays were performed under initial rate conditions (Fig. 3).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Plot of the rate of sulfoxidation of tazarotenic acid versus increasing tazarotenic acid concentration following 30 min incubation of tazarotenic acid with 1 mg/ml pooled human liver microsomes at 37°C. Each data point represents the mean value from four replicates.

	Results

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Incubation Method Optimization. Tazarotenic acid was metabolized by human liver microsomes to one major peak, labeled sulfoxide metabolite (Fig. 2). The Michaelis constant (K_m) for the sulfoxidation of tazarotenic acid, under these conditions, was 24.9 ± 8.0 µM, and the maximum velocity of the reaction (V_max) was 210 ± 38 pmol/mg/min (Fig. 3).

Identification of the Metabolite in Human Liver Microsomes. Preliminary characterization of the major metabolite peak, the sulfoxide metabolite of tazarotenic acid, and parent compound peak (tazarotenic acid) in the incubated human liver microsomal sample was performed by comparing the HPLC retention times with those of the standard sulfoxide metabolite, tazarotenic acid and internal standard as illustrated in Fig. 2. The characterization of the sulfoxide metabolite was also carried out by LC/MS/MS. The mass spectral data of the sulfoxide metabolite were compared with the mass spectral data of standard metabolite (Fig. 4). From the LC/MS/MS spectra of sulfoxide metabolite standard, it was found that this metabolite produced the protonated molecular ion [M + H]⁺ at an orifice voltage of 41 V. 

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Mass spectra of standard sulfoxide metabolite of tazarotenic acid (top panel) and metabolite produced following a 30-min incubation of tazarotenic acid with microsomes containing cDNA-expressed CYP2C8 at 37°C (bottom panel).

Metabolism of Tazarotenic Acid by cDNA-expressed P450 Isozymes. Involvement of CYP2C8 in tazarotenic acid metabolism was determined using cloned human cytochrome P450 isozymes. When tazarotenic acid was incubated with microsomes containing individually expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, or CYP4A11 at 1 mg/ml for 30 min, marked formation of the sulfoxide metabolite was only detected in the microsomes containing CYP2C8 (Fig. 5).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Metabolism of tazarotenic acid to the sulfoxide metabolite by cDNA-expressed P450 isozymes. Tazarotenic acid (10 µM) was incubated with 1 mg/ml microsomal protein for 30 min at 37°C. Each bar represents the mean value from four replicates.

Effect of Chemical Inhibition on CYP2C8-mediated Metabolism of Tazarotenic Acid. Involvement of CYP2C8 in tazarotenic acid metabolism was further confirmed by inhibition of the formation of the sulfoxide metabolite by paclitaxel, 9-cis-retinoic acid, 13-cis-retinoic acid, and quercetin. Paclitaxel, a potential CYP2C8 substrate (Parkinson, 1996) inhibited the formation of the sulfoxide metabolite from tazarotenic acid in a concentration dependent manner with an IC₅₀ of 14.9 ± 6.2 µM (Fig. 6) and a K_i of 30.0 ± 7.0 µM. 9-cis-retinoic acid and 13-cis-retinoic acid, both potential CYP2C8 substrates (Mugford and Kedderis, 1998; Marill et al., 2002) inhibited the formation of the sulfoxide metabolite from tazarotenic acid in a concentration-dependent manner. The IC₅₀ values determined for 9-cis-retinoic acid and 13-cis-retinoic acid were 17.6 ± 2.8 µM and 15.1 ± 2.5 µM, respectively, and their respective K_i values were determined to be 20.2 ± 8.7 and 66.2 ± 14.3 µM. Quercetin, a specific inhibitor of CYP2C8 inhibited the formation of the sulfoxide metabolite from tazarotenic acid in a concentration-dependent manner with an IC₅₀ of 4.07 ± 2.69 µM and a K_i of 19.7 ± 8.2 µM. The results of these chemical inhibition studies are summarized in Table 1.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   IC50 plot illustrating the effect of paclitaxel on the sulfoxidation of tazarotenic acid in pooled human liver microsomes. Control activity is the measured rate of sulfoxidation of tazarotenic acid when 10 µM tazarotenic acid is incubated with 1 mg/ml pooled human liver microsomes, at 37°C for 30 min in the absence of inhibitor. Each data point represents the mean value from four replicates.

Metabolism of Tazarotenic Acid by cDNA-expressed FMO Isozymes. It was determined that both FMO1 and FMO3 could mediate the metabolism of tazarotenic acid to its sulfoxide metabolite using microsomes containing cloned human FMO. When tazarotenic acid was incubated with microsomes expressing FMO1 or FMO3 at 0.25 mg/ml for 30 min there was a marked increase in the formation of the sulfoxide metabolite in both incubations with FMO1 or FMO3 microsomes (Fig. 7). Concurrent to these experiments tazarotenic acid was incubated with CYP3A4 as a negative control.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Metabolism of tazarotenic acid to the sulfoxide metabolite by cDNA-expressed FMO isozymes. Tazarotenic acid (10 µM) was incubated with 0.25 mg/ml microsomal protein for 30 min at 37°C. Incubation of tazarotenic acid with cDNA-expressed CYP3A4 was used as a standard for comparison. Each bar represents the mean value from four replicates.

Effect of Chemical Inhibition on FMO-mediated Metabolism of Tazarotenic Acid. Methimazole is a known substrate of FMO and is commonly used as a competitive inhibitor (Rettie and Fisher, 1999). The involvement of FMO in tazarotenic acid metabolism was supported by the inhibition of sulfoxide metabolite formation by methimazole in a concentration-dependent manner with an IC₅₀ of 60.2 ± 16.2 µM (Fig. 8).

View larger version (20K): [in this window] [in a new window]   Fig. 8.   IC50 plot illustrating the effect of methimazole on the sulfoxidation of tazarotenic acid in pooled human liver microsomes. Control activity is the measured rate of sulfoxidation of tazarotenic acid when 10 µM tazarotenic acid is incubated with 1 mg/ml pooled human liver microsomes, at 37°C for 30 min in the absence of inhibitor. Each data point represents the mean value from four replicates.

	Discussion

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These studies demonstrated that the in vitro metabolism of tazarotenic acid in human liver microsomes results in the production of one major metabolite, namely the sulfoxide metabolite of tazarotenic acid. The metabolite structure was confirmed by LC/MS/MS.

Initially, preliminary correlation analysis studies were conducted using individual liver microsomal preparations from human donors phenotyped by the supplier for the following P450-specific activities: CYP1A2, CYP2A6, CYP2C, CYP2D, CYP2E, CYP3A, and CYP4A. The correlation analysis results demonstrated that correlation coefficient (r) between the tested P450-specific activities with the oxidation of tazarotenic acid to its sulfoxide metabolite was less than 0.6. However, CYP2C19-mediated hydroxylation of S-mephenytoin was used to phenotype CYP2C activity. This probe substrate is specific to CYP2C19 but not to other CYP2C isoforms, namely CYP2C8. Overall, these correlation studies indicated that further experimental work was required to characterize the specific P450 enzymes responsible for the metabolism of tazarotenic acid. Hence, the studies using cDNA-expressed P450 and FMO isoforms and specific inhibitor studies were conducted to determine which metabolic enzymes were responsible for the sulfoxidation of tazarotenic acid.

The specific human microsomal enzymes responsible for the metabolism of tazarotenic acid were identified through incubation with cDNA expressed isozymes. These studies demonstrated that the biotransformation of tazarotenic acid to the sulfoxide metabolite, in human liver microsomes, can be mediated by CYP2C8 and FMO. These findings were supported by inhibition of tazarotenic acid metabolism by specific inhibitors for either CYP2C8 or FMO. Furthermore, CYP2C8 mediation of tazarotenic acid metabolism to the sulfoxide metabolite was supported by the findings of an independent study (Tang-Liu et al., 1999). This study evaluated the effect of tazarotenic acid as an inhibitor of human P450 enzymes. The results of the study concluded that tazarotenic acid most potently inhibited CYP2C8-mediated paclitaxel 6-hydroxylation as compared with other assayed enzymatic activities. Paclitaxel (Taxol) is a drug commonly administered to cancer patients. CYP2C8-mediated metabolism of tazarotenic acid, an acetylenic retinoid, is further supported by a recent study that examined the metabolism of retinoic acid isomers. This study identified CYP2C8 to be active in the metabolism of both 9-cis-retinoic acid and 13-cis-retinoic acid (Marill et al., 2002).

The chemical inhibition studies with CYP2C8 inhibitors determined K_i values of compounds that may be coadministered with tazarotene. The in vivo predicted fractional inhibition (i) value is useful in establishing the potential for interaction between coadministered drugs. The degree of potential competitive inhibition will depend on the substrate concentration, the inhibitor concentration, and the K_m and K_i values (Lin and Lu, 1998). This i value is based on the ratio of the anticipated human plasma concentration of a drug to the K_i of that drug, on the oxidation of tazarotenic acid to its sulfoxide metabolite. Generally, if this ratio is less than 0.01, the likelihood of a significant interaction between the two drugs is low. As listed in Table 1, the predicted fractional inhibition by paclitaxel (Taxol) on tazarotenic acid metabolism to the sulfoxide metabolite is 0.00743 to 0.123 depending on the dose of Taxol administered to the patient. The predicted fractional inhibition by 9-cis-retinoic acid (Panretin) on the sulfoxidation of tazarotenic acid is between 0.00483 and 0.0242. The i value for the potential 13-cis-retinoic acid (Accutane)-tazarotenic acid interaction is between 0.0178 and 0.0474. The potential fractional inhibition on tazarotenic acid metabolism by the dietary flavonoid, quercetin is between 0.00108 and 0.0207. However, the present study demonstrates that the metabolism of tazarotenic acid to the sulfoxide metabolite is mediated by two different enzymes, namely, CYP2C8 and FMO. Potentially, if one metabolic pathway is inhibited the alternative pathway can mediate the metabolism of tazarotenic acid. The present study demonstrated that paclitaxel inhibited the formation of the sulfoxide metabolite of tazarotenic acid in a concentration-dependent manner. At the maximal inhibitor concentration of 1000 µM, 60% control activity was still observed. This residual activity may represent the contribution of other enzymes capable of mediating the sulfoxidation of tazarotenic acid such as FMOs. Furthermore, methimazole also inhibited the sulfoxidation of tazarotenic acid in a concentration-dependent manner. At the maximal inhibitor concentration of 1000 µM, 40% control activity was still observed. In this experiment, the residual activity may be the contribution of enzymes other than FMOs capable of mediating the sulfoxidation of tazarotenic acid such as CYP2C8.

Concomitant administration of two or more drugs can result in undesirable drug-drug interactions. Competitive inhibition of the metabolism of one drug by another may result in undesirable elevations in plasma drug concentrations, which is of clinical importance for both therapeutic and toxicological reasons (Lin and Lu, 1997). Through identifying the enzymes responsible for the metabolism of a drug, drug-drug interactions can be predicted and managed, usually by appropriate dosage adjustment (Lin and Lu, 1997). The results of this study demonstrate that both CYP2C8 and FMO enzymes are responsible for the metabolism of tazarotenic acid in humans.