Introduction

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Olopatadine, (Z)-11-[3-(dimethylamino)propylidene]-6,11-dihydrodibenz[b,e]oxepin-2-acetic acid, is a new histamine H₁ receptor-selective antagonist (Ohshima et al., 1992) that is used in the clinic for the treatment of allergic rhinitis, chronic urticaria, eczema, dermatitis, and conjunctivitis. After oral administration of [¹⁴C]olopatadine to rats and dogs, the main metabolic pathways were 1) N-demethylation to M1 and M2, the N-monodemethyl and N-didemethyl analogs, respectively; 2) hydroxylation of dihydrodibenz[b,e]oxepin ring (M5); and 3) sulfoconjugation of M5 (M4) and N-oxidation (M3) (Ohishi et al., 1995; Fig. 1). After oral administration of olopatadine to human subjects, the metabolites detected in plasma were M1 and M3, but the areas under the plasma concentration-time curve of both M1 and M3 were lower than that of unchanged drug (Fujita et al., 1999). The main elimination pathway of olopatadine in human subjects and animals was via excretion of unchanged drug in urine. Urinary metabolites were mainly M1 and M3, but the amounts of these metabolites were much lower than that of unchanged drug in rat, dog (Ohishi et al., 1995), and human (Tsunoo et al., 1995). Glucuronides of olopatadine and metabolites were not detected in either urine or feces after administration to humans and animals.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Proposed metabolic pathway of olopatadine.

Compounds containing an alkylamino function may be oxidized by cytochrome P450 (P450¹) isozymes with the formation of inhibitory P450-iron (II)-nitrosoalkane metabolite complexes (MICs) (Schenkman et al., 1972; Roberts and Franklin, 1979; Delaforge et al., 1983). Some of these compounds, such as macrolide antibiotics (e.g., erythromycin, troleandomycin; Delaforge et al., 1983; Pessayre et al., 1983), Proadifen (SKF-525A) (Schenkman et al., 1972), and L--acetylmethadol (Roberts and Franklin, 1979), form inhibitory complexes that are stable in vivo. It is hypothesized that this phenomenon is responsible for several problems encountered in patients receiving erythromycin or troleandomycin, such as ergotism in patients receiving concurrent ergot alkaloids and cholestatic jaundice in patients on oral contraceptives (Ludden, 1985). Similarly, tricyclic antidepressants, such as imipramine, desipramine and nortriptyline, inhibit P450 activity by the formation of a MIC (Murray and Field, 1992). Repetitive oral administration of imipramine (100 mg/kg/day for 5 days) caused a decrease in CYP2D1 activity due to MIC formation without changing the content of CYP2D protein (Masubuchi et al., 1995). Thus, the formation of MICs is likely to cause some drug-drug interactions; therefore, it is important to be aware of the possible clinical implications resulting from the formation of MICs.

Olopatadine is a tricyclic drug containing an alkylamino moiety similar to that in imipramine, but there are no published reports investigating MIC formation by olopatadine. Therefore in this study, we have explored the possibility of drug-drug interactions involving olopatadine via MIC formation using in vitro techniques. We have 1) examined the conditions under which olopatadine is N-dealkylated to a primary amine by human liver microsomes (formation of a primary amine is hypothesized to be the first step for MIC formation); 2) identified the enzymes catalyzing olopatadine metabolism; 3) investigated the effects of olopatadine on P450-mediated reactions; and 4) monitored spectral changes that occurred during metabolism of olopatadine by liver microsomes (in general, the formation of MIC exhibits a peak at approximately 454 nm in the absorption spectrum).

	Materials and Methods

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Chemicals. Olopatadine, reference M1, M2, and M3 were synthesized in our institute. [¹⁴C]Olopatadine (2.18 GBq/mmol) was synthesized at Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK) and the purity was more than 99% pure as ascertained by high-performance liquid chromatography (HPLC). Ketoconazole was supplied by Janssen Pharmaceutica (Beerse, Belgium). Other chemicals were obtained from the following sources: 4-acetylaminophenol and N-octylamine (Tokyo Chemical Industries, Tokyo, Japan); bufuralol (BD Gentest, Woburn, MA); S-mephenytoin, 4'-hydroxymephenytoin, and 6-hydroxytestosterone (Sumitomo Chemical Co., Osaka, Japan); chlorzoxazone, desipramine, imipramine, sulfaphenazole, tranylcypromine, and troleandomycin from Sigma-Aldrich (St. Louis, MO); chlorpropamide, corticosterone, furafylline, 1'-hydroxybufuralol, 6-hydroxychlorzoxazone, 4-hydroxytolbutamide, pentoxifylline, and SKF-525A (Sigma/RBI, Natick, MA); and caffeine, diethyldithiocarbamate, phenacetin, phenobarbital, quinidine, testosterone, thiourea, and tolbutamide (Wako Pure Chemicals, Osaka, Japan).

Preparation of Hepatic Microsomal Fractions. Male Wistar rats (7 weeks old) were obtained from Charles River Japan (Kanagawa, Japan) and were held in cages under constant temperature and lighting (12-h light/dark cycle). Rats were killed under ether anesthesia and washed hepatic microsomes were isolated by differential ultracentrifugation (Guengerich et al., 1994). Microsomal fractions were resuspended in 10 mM potassium phosphate, pH 7.4, containing 20% glycerol and 0.1 mM EDTA and were stored at 80°C until required for experiments.

Incubation Conditions. Preliminary results indicated that the rate of formation of M1 and M3 at 10 µM olopatadine was linear at 37°C for an incubation time of up to 1 h and for a microsomal protein concentration up to 2 mg/ml. More than 90% of the initial amount of olopatadine remained after 2-h incubation with 2 mg protein/ml. The concentration of 10 µM olopatadine was chosen for these in vitro incubations because this was the maximum plasma concentration achieved in a phase I clinical study, and also because of a low limit of quantitation for M1 and M3. [¹⁴C]Olopatadine (10 µM, 6.7 kBq/ml final concentration) was incubated with liver microsomes (1 mg protein/ml final protein concentration) in phosphate buffer (100 mM, pH 7.4) at 37°C. Reactions were initiated by the addition of a prewarmed NADPH-generating system (0.8 mM -NADP⁺, 8 mM glucose-6-phosphate, 1 unit/ml glucose-6-phosphate dehydrogenase, and 6 mM MgCl₂), and incubation was continued for up to 2 h. To terminate the reaction at the specified time points, aliquots of the reaction mixtures (100 µl) were mixed with an equal volume of ice-cold acetonitrile containing reference olopatadine, M1, M2, and M3 (100 ng). The samples were centrifuged at 14,020g for 10 min and the supernatant was filtered before carrying out HPLC analysis. The recovery of total radioactivity after the protein-precipitation procedure was calculated to be >95%. For structural elucidation of olopatadine metabolites by HPLC with tandem mass spectrometry (LC/MS/MS), samples were prepared by extracting a large-scale incubation (12.5 ml) using a Bond Elute C₁₈ cartridge (Varian, Harbor City, CA).

Effects of Enzyme Inhibitors and Activators on the Formation of M1 and M3. To determine the effect of compounds on the metabolism of olopatadine, [¹⁴C]olopatadine (10 µM, 6.7 kBq/ml), enhancers or inhibitors, microsomes (1 mg/ml), and NADPH-generating system were mixed and incubated at 37°C for 1 h in a final volume of 100 µl. In the cases of furafylline, diethyldithiocarbamate, troleandomycin, SKF-525A, N-octylamine, and thiourea, the mixture of microsomes and these compounds was preincubated in the presence of a NADPH-generating system at 37°C for 15 min and then the reaction was initiated by addition of [¹⁴C]olopatadine. The concentration of added compounds was 10 µM except for N-octylamine, which was added at 100 µM. All inhibitors were dissolved in methanol; the final concentration of methanol was 1.0 vol% in the reaction mixture.

Effects of Olopatadine on P450 Isozyme-Specific Activities. The effect of olopatadine on the activity of all P450 enzymes was examined using two different incubation methods. A pretreatment method was used to estimate the metabolism-dependent inhibition, e.g., suicidal inhibition, and a simultaneous incubation method was used to assess metabolism-independent inhibition (e.g., competitive inhibition). The following activities were measured for the respective P450 isozymes: phenacetin O-deethylation for CYP1A2 (phenacetin concentration; 100 µM), tolbutamide methylhydroxylation for CYP2C8/9 (tolbutamide concentration; 500 µM), S-mephenytoin 4'-hydroxylation for CYP2C19 (S-mephenytoin concentration; 200 µM), bufuralol 1'-hydroxylation for CYP2D6 (bufuralol concentration; 100 µM), chlorzoxazone 6-hydroxylation for CYP2E1 (chlorzoxazone concentration; 500 µM), and testosterone 6-hydroxylation for CYP3A4 (testosterone concentration; 250 µM). In the preincubation method, incubation of olopatadine was carried out in the presence of the NADPH-generating system at 37°C for 30 min before the addition of the specific probe substrates. In the simultaneous incubation method, olopatadine was added with the specific probe substrates to the microsomal incubation mixture and then the reaction was initiated by addition of the NADPH-generating system. In these studies, higher concentrations of the P450-selective substrates than the respective K_m values were used and, therefore, metabolism of the substrate should not have caused a significant alteration of the reaction rate (Boobis et al., 1998; Pelkonen et al., 1998; Ikeda et al., 2001).

Analytical Methods.

Analysis of Olopatadine and Its Metabolites Olopatadine and its metabolites were separated by HPLC using an instrument purchased from Hitachi (Tokyo, Japan); radioactivity was detected off line with a liquid scintillation counter (Tri-Carb 2200CA or 2700; Packard Instrument Company, Inc., Meriden, CT). Reversed phase chromatography was carried out on a YMC-Pack AM312 (150 × 6.0 mm, 5 µm; YMC Co., Kyoto, Japan) with a mobile phase consisting of 0.1 vol% trifluoroacetic acid in acetonitrile (80:20) at a flow rate of 1 ml/min. The recovery of radioactivity from olopatadine metabolism samples after elution from HPLC was calculated as >95%. The LC/MS/MS analysis of olopatidine and its metabolites was carried out using the method described by Fujita et al. (1999); similar conditions were also used for the structural elucidation of olopatadine metabolites by LC/MS/MS.

Analysis of Marker Metabolites for Specific P450 Substrates. The measurement of P450 activity involved the following methods with some minor modifications: phenacetin O-deethylase according to Kajita et al. (2000); tolbutamide methylhydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, and testosterone 6-hydroxylation according to Newton et al. (1995); and S-mephenytoin 4'-hydroxylation according to Meier et al. (1985). The HPLC instrumentation used for measurement of P450 activities consisted of an LC10A pump system (Shimadzu, Kyoto, Japan), a UV detector (SPD-10A; Shimadzu), and a fluorescence detector (L-7480; Hitachi).

Spectral Analysis. The formation of an MIC was studied by monitoring spectral changes that occurred during incubations with rat liver microsomes. The reaction mixture contained phosphate buffer (100 mM, pH 7.4), 1 mM EDTA, 10 mM MgCl₂, and 1 mg of microsomal protein at 37°C. Olopatadine, M1, imipramine, or desipramine, dissolved in ethanol, were added to the test cuvette, and the same volume of ethanol was added to the reference cuvette. After the baseline was corrected, the NADPH-generating system was added to both cuvettes and the final volume adjusted to 1 ml, and then the difference spectra from 380 to 500 nm were recorded during incubation at 37°C at the designated time points using a 150-20 spectrophotometer (Hitachi).

	Results

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Olopatadine Metabolism. Two different metabolites, M1 and M3, were formed when olopatadine was incubated with human liver microsomes in the presence of an NADPH-generating system: after 1-h incubation, M1 and M3 accounted for 5.2 and 30.5% of the initial olopatadine, respectively. Structural information for these metabolites was obtained by LC/MS/MS analysis with electrospray ionization (ESI). The protonated molecular ion [M + H]⁺ of M1 was observed at m/z 324 in the positive ESI spectrum, and fragment ions were detected at m/z 247 and 165 (Fig. 2A). The protonated molecular ion [M + H]⁺ of M3 was observed at m/z 354 in the positive ESI spectrum, and fragment ions were recorded at m/z 247 and 165 (Fig. 2B). The protonated molecular ion [M + H]⁺ of olopatadine was observed at m/z 338 in the positive ESI spectrum, and fragment ions were detected at m/z 247 and 165 (Fig. 2C). From these data, M1 and M3 were identified tentatively as N-monodemethylolopatadine and olopatadine N-oxide, respectively, and this assignment was confirmed by demonstrating that these metabolites had identical retention times and mass spectra to the respective authentic standards. The formation of both M1 and M3 by human liver microsomes was found to be NADPH-dependent, and the formation rate of M1 and M3 was 0.330 and 2.50 pmol/min/mg protein, respectively. Kinetic parameters could not be calculated for the metabolites, because only low amounts of the metabolites were formed. In addition, M3 was reduced to olopatadine during incubation with human liver microsomes in the presence of NADPH under aerobic condition (data not shown). Therefore, the kinetic parameters could not be calculated accurately.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Mass spectra of M1 (A), M3 (B), and olopatadine (C).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effects of various compounds on M1 () and M3 () formation by human liver microsomes. Liver microsomes (1 mg of protein) were incubated for 60 min at 37°C with olopatadine and other specified compounds in the presence of an NADPH-generating system in a final volume 0.2 ml. For metabolism-based inhibitors or enhancers (i.e., SKF-525A, furafylline, diethyldithiocabamate, troleandomycin, and N-octylamine), the microsomes were preincubated with these compounds for 15 min at 37°C in the presence of an NADPH-generating system before the addition of olopatadine. The concentrations of added inhibitors were 10 µM except for N-octylamine, which was added at 100 µM. The column labeled "Heat-treatment" represents the result obtained when the microsomes were heated at 45°C for 2 min. Activities are expressed as a percentage relative to the control experiment. The control activities of M1 and M3 formation were 0.330 and 2.50 pmol/min/mg protein, respectively. Each column represents the mean of two incubations. N.D., not detected.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Formation of M1 () and M3 () by microsomes from cells expressing the specified recombinant P450 and FMO isozymes. Cell microsomes of recombinant P450 and FMO isozymes were incubated at 37°C with olopatadine in the presence of an NADPH-generating system. Each column represents the mean of two incubations.

Effects of Olopatadine, M1, and M3 on P450 Activities. Simultaneous addition of substrate and olopatadine (100 µM) did not affect the activities of CYP1A2 (phenacetin O-deethylation), CYP2C8/9 (tolbutamide methylhydroxylation), CYP2C19 (S-mephenytoin 4'-hydroxylation), CYP2D6 (bufuralol 1'-hydroxylation), CYP2E1 (chlorzoxazone 6-hydroxylation), and CYP3A4 (testosterone 6-hydroxylation) (Fig. 5). However, specific P450 inhibitors inhibited the relevant P450 enzyme-selective activity under the incubation conditions used (Fig. 5).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Effects of olopatadine (closed symbol) and selective inhibitors (open symbol) on P450 enzyme-specific activities in human liver microsomes. Liver microsomes were incubated at 37°C with olopatadine or a specific substrate of P450 enzymes in the presence of an NADPH-generating system. Activities are expressed as a percentage relative to the control experiment. The control activities of phenacetin O-deethylation, tolbutamide methylhydroxylation, S-mephenytoin 4'-hydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, and testosterone 6-hydroxylation were 528, 65.7, 37.7, 82.0, 1080, and 3150 pmol/min/mg protein, respectively. Values are the mean of two incubations.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Effects of metabolite intermediate complexation with olopatadine, M1, imipramine, and desipramine on P450 enzyme-specific activities in human liver microsomes. Incubation mixtures containing olopatadine, M1, imipramine, or desipramine were preincubated in the presence of an NADPH-generating system at 37°C for 30 min, and the reaction was initiated by addition of substrate. Activities are expressed as a percentage relative to a control incubation performed in the absence of test compounds. The control activities of phenacetin O-deethylation, tolbutamide methylhydroxylation, S-mephenytoin 4'-hydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, and testosterone 6-hydroxylation were 164, 54.7, 40.5, 70.0, 723, and 3176 pmol/min/mg protein, respectively. Values are the mean of two incubations.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Spectra obtained during incubations of olopatadine (A), M1 (B), imipramine (C), and desipramine (D) with P450 in rat liver microsomes. Optical difference spectra were obtained 5, 10, 15, and 30 min after the addition of an NADPH-generating system. For details, see the text under Materials and Methods.

	Discussion

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Many alkylamine derivatives are converted to reactive species that produce inhibitory complexes with P450 isozymes during microsomal P450-mediated metabolism. Because the first report of this phenomenon with SKF-525A (Schenkman et al., 1972), it has been demonstrated that amphetamine, macrolide antibiotics such as erythromycin and troleandomycin (Delaforge et al., 1983; Pessayre et al., 1983), and antihistamines such as diphenhydramine and orphenadrine also form complexes with P450 isozymes (Bast and Noordhoek, 1982; Bast et al., 1984). Similarly tricyclic antidepressants, such as imipramine, desipramine, and nortriptyline, inhibit P450 activity by the formation of an MIC (Murray and Field, 1992). Recent evidence also suggests that pharmacokinetic drug-drug interactions with clinical significance may be due, in part, to MIC of the inhibitor with P450 isozymes causing a decrease of P450 activity. The inhibitory effect seems to be relatively selective for the corresponding P450 isozyme(s); for example, clarithromycin-mediated MIC seems to be restricted to CYP3A4 in human liver (Mayhew et al., 2000). It has been confirmed that clarithromycin reduces the elimination of drugs metabolized by CYP3A, such as midazolam (Gorski et al., 1998), omeprazole (Furuta et al., 1999), and cisapride (van Haarst AD et al., 1998; Piquette, 1999) in vivo. Because olopatadine, like imipramine, contains an alkylamino moiety there was a possibility that it also might inhibit P450 activities via the formation of MIC. Therefore, we investigated the effects of olopatadine on P450 activities in human liver microsomes to assess the potential for drug-drug interactions between olopatadine and other drugs.

When olopatadine was metabolized by human liver microsomes in the presence of a NADPH-generating system, two metabolites were formed: N-monodemethylolopatadine M1 and an N-oxide M3. It is noteworthy that N-demethylation seems to be an important first step for the formation of MIC (Bensoussan et al., 1995). However, only low amounts of M1 were generated from olopatadine in human liver microsomes. After oral administration to human subjects, the major metabolites of olopatadine in plasma and urine were reported to be M1 and M3, but the concentrations of these metabolites were much lower than that of unchanged drug (Tsunoo et al., 1995; Fujita et al., 1999). Thus, the low level of olopatadine metabolism observed in vitro is consistent with the low amount of olopatadine metabolites formed in vivo.

N-Dealkylation can be catalyzed by different P450 isozymes. For example, N-dealkylation of several drugs, including alfentanil (Yun et al., 1992) and amiodarone (Trivier et al., 1993), is catalyzed by CYP3A isozymes in human; a limited number of drugs, including amiflamine (Alvan et al., 1984) and citalopram (Gram et al., 1993) are N-dealkylated by CYP2D6; other isozymes that catalyze N-dealkylation in human are CYP1A2 (caffeine; Butler et al., 1989) and CYP2C19 (diazepam; Bertilsson et al., 1989).

In addition, drugs containing nitrogen can be metabolized via N-oxidation. The oxidation of nitrogen atoms is often catalyzed by P450 isozymes and FMO. In the present study, two approaches were used to identify the drug-metabolizing enzymes responsible for the formation of M1 and M3, namely, chemical inhibition and formation by cDNA-expressing human enzymes. Troleandomycin and ketoconazole, which are both selective inhibitors of CYP3A (Newton et al., 1995), significantly inhibited M1 formation, but other inhibitors of P450 isozymes did not inhibit. Also, expressed CYP3A4 exhibited the highest activity for M1 formation, which was over 5-fold higher than the activity of other P450 isozymes; M1 was not generated by FMO isozymes. Therefore, it was concluded that M1 formation was mainly catalyzed by CYP3A.

On the other hand, M3 formation was enhanced by N-octylamine and inhibited by thiourea, but not inhibited by other inhibitors of P450 isozymes. N-octylamine is an activator of FMO (McManus et al., 1987) and an inhibitor of P450 (Hodgson and Levi, 1998), whereas thiourea is an inhibitor of FMO (Cashman et al., 1993) and P450 (Ortiz de Montellano and Correia, 1995). These results suggest that the major metabolizing enzymes involved in M3 formation are FMO isozymes. This is further substantiated by the observation that expressed FMO1 and FMO3 exhibited a higher activity of catalyzing the formation of M3 than the P450 enzymes.

In addition, we investigated whether olopatadine might affect P450-dependent metabolism of other drugs. In general, P450 inhibitors can be divided into three categories according to the mechanism: 1) agents that bind to P450s reversibly; 2) agents that form quasi-irreversible complexes with the heme iron atom of P450s; and 3) agents that bind irreversibly to the protein or the prosthetic heme group, or that accelerate degradation of the prosthetic heme group. In this study, three approaches were used to estimate the inhibitory effect of olopatadine on P450 activities, namely, the effects on P450 activities using two different incubation methods and a spectroscopic study. The two different incubation methods involved 1) preincubation and 2) simultaneous incubation with olopatadine and human liver microsomes in the presence of an NADPH-generating system. Metabolism-based inhibitors, such as furafylline and sorivudine (Ito et al., 1998), irreversibly bind to the enzyme, forming an MIC, and reduce both the activity and amount of the target enzymes. Thus, when metabolism-based inhibitors are incubated with target enzymes plus relevant cofactors before the addition of substrates, enzyme activity is inhibited strongly. On the other hand, when reversible (competitive) inhibitors are incubated simultaneously with target enzyme, cofactors, and substrates, enzyme activities are moderately inhibited. Because olopatadine and M1 did not inhibit P450 activities under either of the incubation conditions used, it was concluded that the compounds were not important inhibitors of P450 enzymes.

The above-mentioned conclusion was supported by the finding that M1 did not produce a change in the optical difference spectrum in rat liver microsomes, although a positive control, desipramine, did produce a time-dependent change. When desipramine was incubated with rat liver microsomes, the spectrum shifted clearly at ~454 nm, which was presumed to be due to the formation of an MIC. However, neither N-dealkylated olopatadine, M1, nor olopatadine itself caused a change in the optical difference spectrum in rat liver microsomes. These results suggest that the primary amine M1 does not form an MIC.

In conclusion, the present study indicates that olopatadine has no inhibitory effects on drug metabolism involving CYP1A2, CYP2C8/9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Lack of any inhibitory effects is probably not surprising because olopatadine is poorly metabolized both in vitro and in vivo; the main elimination pathway is via urinary excretion of unchanged compound. Because the metabolic clearance of olopatadine in humans is very low, concurrent administration of inhibitors of metabolism is unlikely to alter the pharmacokinetics of olopatadine significantly. Therefore, we conclude that drug-drug metabolic interactions involving MIC phenomenon by olopatadine are unlikely in clinical use.