Introduction

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Loratadine (LOR¹) is an orally effective, nonsedating, long-acting H1 receptor antagonist, marketed world wide as Claritin, with no autonomic anticholinergic effects in humans (Bedard et al., 1985; Batenhorst et al., 1986; Villani et al., 1986). Metabolic studies in humans (Katchen et al., 1985; Hilbert et al., 1987; Simons and Simons, 1999) have established that loratadine is rapidly absorbed and undergoes extensive first-pass metabolism to descarboethoxyloratadine (desloratadine, DL). DL is also pharmacologically active and is present in the plasma at low concentrations due to metabolism to several hydroxylated metabolites, including the active metabolite, 3-hydroxydesloratadine (3-OH-DL), which are excreted as conjugates (Katchen et al., 1985). Yumibe et al. (1995) identified CYP2D6 and CYP3A4 as the primary human liver enzymes responsible for the metabolism of LOR to DL with a K_m range for LOR of 7 to 35 µM. Loratadine is 97 to 99% plasma protein bound with an apparent oral clearance of 142.0 ± 56.5 ml/min/kg for a 40-mg dose (Hilbert et al., 1987). The clinical plasma C_max for a 10-mg dose is 4.7 ± 2.7 ng of LOR/ml (Hilbert et al., 1987). For DL and 3-OH-DL, the clinical plasma C_max for a 10-mg dose are 4.0 and 2.8 ng/ml, respectively (Hilbert et al., 1987; personal communication Dr. Samir Gupta, Schering-Plough Research Institute). The purpose of this study was to assess the potential of interactions of LOR, DL, and 3-OH-DL with concomitantly administered drugs. The inhibition profile of LOR, DL, and 3-OH-DL was characterized toward five human cytochrome P-450 enzymes: CYP1A2 (7-ethoxyresorufin O-deethylation), CYP2C9 (diclofenac 4'-hydroxylation), CYP2D6 (dextromethorphan O-demethylation), CYP2C19 (mephenytoin 4'-hydroxylation), and CYP3A4 (testosterone 6-hydroxylation and dextromethorphan N-demethylation) using human liver microsomes pooled from 15 individuals.

	Materials and Methods

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Chemicals and Microsomes. Ketoconazole, 11-hydroxytestosterone, and S-(+)-mephenytoin were purchased from Ultrafine Chemicals (Manchester, England). Levallorphan was purchased from Research Biochemicals International (Natick, MA). Resorufin was purchased from Fluka (Milwaukee, WI), and -naphthoflavone was purchased from Aldrich (Milwaukee, WI). Loratadine, desloratadine, and 3-OH-desloratadine were obtained from the Research Activities Stockroom of Schering-Plough Research Institute. Pooled human liver microsomes were purchased from XenoTech LLC (Kansas City, KS). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO).

Enzyme Specific Assays. 7-Ethoxyresorufin O-deethylation was determined using the fluorometric method of Dutton and Parkinson (1989) with the following modifications: microsomes (1.2 mg/ml) were incubated for 5 min at 37°C in 50 mM potassium phosphate buffer (pH 7.4), 1.5 units/ml G6Pdh, 5 µM 7-ethoxyresorufin, and the components listed in the reference. -Naphthoflavone (50 µM) was the positive inhibitor control. The relative amount (pmol/min/mg) of resorufin in the supernatant was measured using a Hitachi F-2000 spectrofluorometer (_ex: 535 nm; _em: 585 nm). The dextromethorphan assay, which measures O-demethylase activity (CYP2D6) and N-demethylase activity (CYP3A4), was determined by modification of the methods of Kronbach et al. (1987), Kronbach (1991), and Chen et al. (1990). Briefly, microsomes (1 mg/ml) were incubated for 10 min at 37°C with 50 mM potassium phosphate buffer (pH 7.4), 3 mM MgCl₂, 1 mM EDTA, dextromethorphan (20 µM for CYP2D6; 300 µM for CYP3A4), 1 mM -NADP, 5 mM G6P, and 1.5 units/ml G6Pdh. Ketoconazole (2 µM) was the positive inhibitor control for CYP3A4; quinidine (2 µM) was the positive inhibitor control for CYP2D6. The reaction was terminated with 30% acetic acid containing levallorphan (internal standard). Protein was precipitated by centrifugation, and the supernatant was injected into a Waters HPLC equipped with a Shimadzu RF-535 fluorescence detector (_ex: 280 nm; _em: 310 nm) and a Microsorb-MV phenyl column. The flow rate was 0.75 ml/min with a run time of 25 min. The data (peak area ratio) were collected and analyzed using Waters Expert Ease software, version 860/V3.2. Diclofenac 4'-hydroxylation was determined by the method of Leemann et al. (1993) with the following modifications: microsomes (0.1 mg/ml) was incubated for 10 min at 37°C in 50 mM potassium phosphate buffer (pH 7.4) with 5 mM MgCl₂, 10 µM diclofenac, 25 units/ml G6Pdh, 1 mM -NADP, and 10 mM G6P. Sulfaphenazole (20 µM) was the positive inhibitor control. The supernatant was injected onto a Waters 2690 Alliance HPLC system equipped with a Waters 996 photodiode array detector and a Zorbax RX-C8 (4.6 × 250 mm) column. The flow rate was 1 ml/min and the run time was 20 min. The mobile phase consisted of 50% acetonitrile with 1% acetic acid and 50% 20 mM ammonium acetate, pH 4.0. The data (peak area) were collected at a wavelength of 267 nm and analyzed using Waters Millennium software, version 3.05.01. S-(+)-Mephenytoin 4'-hydroxylation was determined using the following modifications of Meier et al. (1985) and Wrighton et al. (1993). Microsomes (2 mg/ml) were incubated for 30 min at 37°C with 0.5 M HEPES buffer (pH 7.4), 5 mM MgCl₂, 100 µM S-(+)-mephenytoin, 15 units/ml G6Pdh, 1 mM -NADP, and 10 mM G6P. The positive inhibitor control, 17-hydroxyprogesterone (Yamazaki and Shimada, 1997), was run at 20 µM. Ice-cold acetonitrile terminated the reaction, and the protein was precipitated by centrifugation. The supernatant was injected into a Waters 2690 Alliance HPLC System equipped with a Waters 996 photodiode array detector and a Zorbax RX-C8 (4.6 × 250 mm) column. The flow rate was 1 ml/min and the run time was 20 min. The mobile phase consisted of 29% acetonitrile with 1% acetic acid and 71% 20 mM ammonium acetate, pH 4.0. The data (peak area) were collected at a wavelength of 224 nm and analyzed using Waters Millennium software, version 3.05.01. The 6-hydroxylation of testosterone was determined by a modification of the method of Soderfan et al. (1987). Microsomes (0.24 mg/ml) were incubated for 10 min at 37°C with 50 mM potassium phosphate buffer, 3.3 mM MgCl₂, 200 µM testosterone, and 1 mM NADPH. Ketoconazole (2 µM) was the positive inhibitor control. The reaction was terminated with acetonitrile containing 11-hydroxytestosterone (internal standard). The protein was precipitated by centrifugation. The supernatant was injected onto a Waters 2690 Alliance HPLC system equipped with a Waters 996 photodiode array detector (245-nm wavelength) and a Zorbax RX-C8 (4.6 × 150 mm) column maintained at 40°C. The flow rate was 1 ml/min and the run time was 30 min. The mobile phase consisted of a 20 mM ammonium acetate and methanol gradient system. Initial conditions were 45% ammonium acetate for 15 min followed by ramping to 100% methanol at 30 min. The data (peak area ratio) were collected and analyzed using Waters Millennium software, version 3.05.01.

	Results and Discussion

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As shown in Table 1, LOR, DL, and 3-OH-DL did not inhibit the CYP1A2-mediated O-deethylation of 7-ethoxyresorufin at concentrations up to 10 µM. LOR inhibited the CYP2C9 catalyzed 4'-hydroxylation of diclofenac by 31% at 10 µM (3829 ng/ml); this is approximately 815 times the maximal steady-state plasma concentration (C_max 4.7 ± 2.7 ng/ml; Hilbert et al., 1987) expected in humans at a therapeutic dose of 10 mg of LOR per day. 3-OH-DL caused 10% inhibition of CYP2C9 activity and DL no inhibition at a 10 µM concentration. LOR at 10 µM inhibited the CYP2D6 catalyzed O-demethylation of dextromethorphan by 55%. The IC₅₀ was estimated to be 8.1 µM (3100 ng of LOR/ml) (Fig. 1A), which is approximately 660 times the expected therapeutic LOR plasma exposure concentrations. The K_i for the CYP2D6 reaction was estimated to be 2.7 µM (1034 ng of LOR/ml) (Fig. 1B). DL at 10 µM (3108 ng of DL/ml) inhibited the CYP2D6 catalyzed reaction by 14% (approximately one-fourth of the LOR inhibition of CYP2D6, and 777 times greater than the DL (10-mg dose) plasma C_max of 4.0 ± 1.7 ng/ml; Hilbert et al., 1987). 3-OH-DL inhibited CYP2D6 activity by 2% at 10 µM (3268 ng of 3-OH-DL/ml) concentrations (this concentration is more than 1167 times those expected for expected 3-OH-DL plasma C_max of 2.8 ng/ml; personal communication Dr. Samir Gupta, Schering-Plough Research Institute). LOR at 10 µM inhibited the 4'-hydroxylation of S-(+)-mephenytoin by 99%. The IC₅₀ was estimated to be 0.76 µM (291 ng/ml) (Fig. 1C), which is approximately 62 times the expected therapeutic LOR plasma exposure concentrations. The K_i of the CYP2C19 reaction was estimated to be 0.61 µM (234 ng/ml) LOR (Fig. 1D). DL and 3-OH-DL did not significantly inhibit CYP2C19-catalyzed reaction at concentrations of 10 µM. LOR, DL, and 3-OH-DL inhibited the CYP3A4 catalyzed metabolism of testosterone by 13, 20, and 10%, respectively, at 10 µM. CYP3A4-mediated dextromethorphan N-demethylation was not inhibited to any extent by LOR and was inhibited 16 and 25% by DL and 3-OH-DL, respectively, at 10 µM. In conclusion, LOR moderately inhibited CYP2C19 and CYP2D6 activity, which is in accordance with earlier work performed by Nicolas et al. (1999). This inhibition is 62 to 660 times the maximal single 10-mg dose plasma concentration. DL and 3-OH-DL demonstrated little or no inhibition potential for the five cytochrome P-450 enzymes investigated. These results indicate that LOR and the active metabolites DL and 3-OH-DL at therapeutic concentrations are unlikely to affect the oxidative metabolism and intrinsic clearance of coadministered drugs, which are metabolized by CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP3A4.

View this table: [in this window] [in a new window]   TABLE 1 Effects of loratadine, desloratadine, and 3-OH-desloratadine on CYP activities in human hepatic microsomes The IC50 experiments were run at approximate Km values established in our laboratory for pooled human liver microsomes and, in general, correspond with Km values found in the literature: [3A4-dextromethorphan] Schmider et al., 1997; [3A4-testosterone] Wang et al., 1997; [1A2] Lin et al., 2001; [2C19] Easterbrook et al., 2001; [2C9] Tang et al., 2000; [2D6] Hickman et al., 1998.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Kinetic analysis graphs of loratadine. A, estimation of LOR IC50 for dextromethorphan O-demethylation (CYP2D6). Quinidine, the positive inhibitor control, produced 95% inhibition of this enzyme at 2 µM. B, Ki determination for the effect of LOR on O-demethylation of dextromethorphan (CYP2D6). The 30 µM LOR value at the 5 µM dextromethorphan concentration was not included due to a large variance in duplicate samples. Equations of the lines: 5 µM, y = 0.6448x + 3.6782; 20 µM, y = 0.1587x + 1.4923; 80 µM, y = 0.034x + 0.7832. C, estimation of LOR IC50 for S-(+)-mephenytoin 4'-hydroxylation (CYP2C19). The positive inhibitor control, 17-hydroxyprogesterone (20 µM), inhibited the formation of the 4'-hydroxylated product by 50%. D, Ki determination for the effect of LOR on 4'-hydroxylation of S-(+)-mephenytoin (CYP2C19). Equations of the lines: 30 µM, y = 2E05x + 1E05; 90 µM, y = 4E06x + 4E06; 270 µM, y = 2E06x + 2E06. The estimated IC50 values were determined with nonlinear regression analysis: y = Imax + (Vo  Imax) · S/(IC50 + S) using GraphPad Prism 2.01 software (San Diego, CA). Where y = activity (% of control), Imax= maximum inhibition, Vo= control activity, S = concentration of test compound, and IC50 = concentration at half-maximal enzyme inhibition. The estimated Ki values were determined with Dixon Plot analysis using Microsoft Excel 97 SR-2 to fit the curves using linear regression.