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CYTOCHROME P450 3A4 IS THE MAJOR ENZYME RESPONSIBLE FOR THE METABOLISM OF LAQUINIMOD, A NOVEL IMMUNOMODULATOR

Preclinical Development, Active Biotech Research AB, Lund, Sweden (H.T., I.H., R.P., B.S., P.O.G.); Astra Zeneca R&D, Lund, Sweden (J.S.); and Department of Cell and Molecular Biology, Lund University, Lund, Sweden (H.T., J.S.)

(Received September 9, 2004; accepted March 4, 2005)

	Abstract

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In the present study, the involvement of cytochrome P450 enzyme(s) in the primary metabolism of laquinimod, a new orally active immunomodulator, has been investigated in human liver microsomes. Hydroxylated and dealkylated metabolites were formed. The metabolite formation exhibited single enzyme Michaelis-Menten kinetics with apparent K_M in the range of 0.09 to 1.9 mM and V_max from 22 to 120 pmol/mg/min. A strong correlation between the formation rate of metabolites and 6ß-hydroxylation of testosterone was obtained within a panel of liver microsomes from 15 individuals (r² = 0.6 to 0.94). Moreover, ketoconazole and troleandomycin, specific inhibitors of CYP3A4 metabolism, demonstrated a significant inhibition of laquinimod metabolism. Furthermore, in incubations with recombinant CYP3A4, all the primary metabolites were formed. In vitro interaction studies with CYP3A4 substrates and possible concomitant medication demonstrated that laquinimod inhibits the metabolism of ethinyl estradiol with an IC₅₀ value of about 150 µM, which is high above the plasma level of laquinimod after clinically relevant doses. Ketoconazole, troleandomycin, erythromycin, prednisolone, and ethinyl estradiol inhibited the metabolism of laquinimod, and IC₅₀ values of 0.2, 11, 24, 87, and 235 µM, respectively, were calculated. In conclusion, the present study demonstrates that laquinimod is a low affinity substrate for CYP3A4 in human liver microsomes. The likelihood for in vivo effects of laquinimod on the metabolism of other CYP3A4 substrates is minor. However, inhibitory effects on the metabolism of laquinimod by potent and specific inhibitors of CYP3A4, such as ketoconazole, are anticipated and should be considered in the continued clinical program for laquinimod.

The pharmacokinetic properties of laquinimod have been studied in several preclinical species used for the pharmacological and toxicological evaluation of the compound. In mouse, rat, rabbit, and dog, laquinimod pharmacokinetics is characterized by a high oral bioavailability, a low total clearance, and a small volume of distribution (unpublished results). Furthermore, the compound seems to be eliminated by metabolism, and both hydroxylated and demethylated products have been detected in urine from all the studied species. Hydroxylation and demethylation are reactions known to be catalyzed by the cytochrome P450 (P450) enzymes (Nelson et al., 1996). This large family of enzymes, bound to the endoplasmic reticulum, catalyzes a variety of reactions of xenobiotic metabolism as well as metabolism of endogenous compounds.

In the present study, the primary metabolism of laquinimod was investigated using different enzymatic preparations including human liver microsomes and recombinantly expressed enzymes. In addition, in vitro studies were performed to predict possible drug-drug interactions of laquinimod in humans.

	Materials and Methods

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Chemicals. [¹⁴C]Laquinimod (batch rd-A, with a specific radioactivity of 56.0 mCi/mmol and a radiochemical purity >95%) and the nonradioactive reference compounds, laquinimod, ABR-215791 (M1), ABR-218287 (M3), ABR-218373 (M4), ABR-215818 (M5), and ABR-215174 (M6) were synthesized at Active Biotech Research AB (Lund, Sweden). [³H]Ethinyl estradiol (specific radioactivity 52.3 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). NADPH, coumarin, diclofenac, quinidine, sulfaphenazole, troleandomycin, erythromycin, ethinyl estradiol, and prednisolone were purchased from Sigma-Aldrich (St. Louis, MO). Chlorzoxazone, furafylline, and ketoconazole were obtained from Sigma/RBI (Natick, MA). All other chemicals were of analytical grade.

Enzyme Systems. Pooled human liver microsomes and a panel of liver microsomal samples from 15 individual livers (Reaction Phenotyping Kit) were obtained from XenoTech (Lenexa, KS).

The human microsomes had been characterized with respect to total P450 content and specific content of individual P450 enzymes, which were determined from their catalytic activity for the biotransformation of the following substrates: CYP1A2 (7-ethoxyresorufin O-dealkylation), CYP2A6 (coumarin 7-hydroxylation), CYP2B6 (7-ethoxy-trifluoromethylcoumarin O-deethylation), CYP2C8 (paclitaxel 6-hydroxylation), CYP2C9 (tolbutamide methylhydroxylation), CYP2C19 (S-mephenytoin 4-hydroxylation), CYP2D6 (dextromethorphan O-demethylation), CYP2E1 (chlorzoxazone 6-hydroxylation), and CYP3A4/5 (testosterone 6ß-hydroxylation). cDNA-expressed human CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were obtained from Gentest (Woburn, MA).

Assay Conditions. Incubations were performed with laquinimod (5-3000 µM) and 1 to 4 mg/ml microsomal protein in 50 mM sodium phosphate buffer, pH 7.4, at 37°C for 0 to 60 min. Preliminary experiments were performed to establish conditions for reasonable substrate consumption and time and protein linearity of metabolite formation. The incubation mixture was preincubated for 5 min before addition of NADPH (10 mM, final concentration). The reaction was stopped by addition of an equal volume of ice-cold acetone and centrifuged at 10,000g for 10 min. The supernatant was transferred to autosample vials and analyzed by high performance liquid chromatography (HPLC). For identification of metabolites, incubations were performed with laquinimod (1500 µM) and 2 mg of microsomal protein for 60 min.

In the correlation studies, the metabolism of laquinimod was determined across 15 different human liver microsomal preparations, which had been characterized for specific P450 substrate activities. To give an appropriate evaluation of the metabolism for this correlation study, 300 µM laquinimod was incubated with 3 mg/ml microsomal protein.

In the inhibition studies, incubations were performed with human liver microsomes and specific inhibitors of P450 enzymes. Laquinimod, 200 µM, was incubated in 3 mg/ml microsomal protein and one of the following enzyme inhibitors: 20 µM furafylline (Kunze and Trager, 1993), 25 µM coumarin (Pearce et al., 1992), 200 µM diclofenac (Transon et al., 1996), 50 µM sulfaphenazole (Relling et al., 1990), 25 µM quinidine (Inaba et al., 1985), 50 µM chlorzoxazone (Peter et al., 1990), 2 µM ketoconazole (Baldwin et al., 1995), or 100 µM troleandomycin (Guengerich, 1990) at pH 7.4 for 20 min at 37°C. Each inhibitor was added in ethanol (final concentration of ethanol in each incubation was 2%). Control experiments with 2% ethanol and control experiments without NADPH were performed in parallel. All samples were made in duplicate. There was a preincubation equilibrium and inactivation period for 10 min at 37°C, with inhibitor and NADPH, for the mechanism-based inhibitors furafylline and troleandomycin. The other incubation mixtures were preincubated for 5 min at 37°C before addition of NADPH (10 mM, final concentration).

The metabolism of laquinimod by cDNA-expressed P450 enzymes was performed by incubating laquinimod (100 µM) with microsomes containing one of the following expressed P450 isoforms from insect cells: CYP1A1, 1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 (1-2 mg of microsomal protein/ml). Microsomes from nontransfected cells were used as controls. Laquinimod metabolism in expressed CYP3A4 was next evaluated over a range of substrate concentrations (5-3000 µM) and kinetic parameters for formation of metabolites were obtained.

To study the effect of laquinimod on the metabolism of ethinyl estradiol and prednisolone, incubations were performed with ethinyl estradiol (100 µM) or prednisolone (100 µM) and laquinimod at a concentration range of 5 to 3000 µM, and the IC₅₀ values were calculated. Preliminary experiments were performed to establish conditions for reasonable substrate consumption and time and protein linearity of metabolite formation from ethinyl estradiol and prednisolone.

To determine the effect of known CYP3A4 substrates on the metabolism of laquinimod, the compound (200 µM) was incubated with one of the following compounds: ketoconazole (0.01-20 µM), troleandomycin (0.5-500 µM), erythromycin (1-750 µM), prednisolone (1-1000 µM), or ethinyl estradiol (1-400 µM), with 3 mg/ml protein for 10 to 20 min, and IC₅₀ values were calculated. Concentrations of the different CYP3A4 substrates were chosen based on data from the literature and preliminary experiments to determine a concentration range for each compound, covering no inhibition to almost complete inhibition. The mechanism-based inhibitors troleandomycin and erythromycin were preincubated for 10 min at 37°C with NADPH before addition of laquinimod.

HPLC Analyses. Quantification of laquinimod and its metabolites was obtained with an HPLC system. The samples were injected (50-100 µl) into the chromatographic system and separated on a reversed phase Symmetry C18 column (5 µm, 3.9 x 150 mm) with a reversed phase Symmetry C18 guard column (Waters, Milford, MA) and a linear gradient system from 20:10:70 to 90:10:0 (acetonitrile/1 M phosphoric acid/water). The analyses were performed at room temperature at a flow of 1.0 ml/min. Radioactivity was detected by a flow detector (Flo-One ß; PerkinElmer Life and Analytical Sciences), with a 0.5-ml flow cell. The mobile phase was mixed postcolumn with Ultima-Flo AP (PerkinElmer Life and Analytical Sciences) at a flow of 3 ml/min.

Ethinyl estradiol and metabolites were quantified using an HPLC system (Fernandez et al., 1993) and radiochemical detection. Prednisolone and metabolites were quantified by UV detection at 254 nm. Prednisolone was analyzed by HPLC with an isocratic system of 25% tetrahydrofuran in water.

Calculations and Kinetic Analysis. The metabolite peak areas from HPLC analysis were converted into picomoles and expressed in relation to milligrams of protein per minute or nanomoles of P450 per minute.

The nature of laquinimod microsomal metabolism was characterized graphically by Eadie-Hofstee plots for the different metabolites and the kinetic parameters K_M, V_max, and intrinsic clearance (CL_int) were estimated by nonlinear regression of the saturation curve by the nonlinear procedure PROC NLIN in the SAS statistical software (SAS/STAT; SAS Institute Inc., 1989).

CL_int was estimated as V_max/K_M using either the one-site or the two-site Michaelis-Menten model: V = V_max · S/(K_M + S) + CL₂ · S. Correlation coefficients (r²) were calculated using linear regression analysis.

Formation of metabolites was plotted versus inhibitor concentration. The IC₅₀ values were calculated by nonlinear regression of the plot of metabolite formation versus inhibitor concentration, using the one-site competition model in Prism, version 2.0 (GraphPad Software Inc., San Diego, CA).

	Results

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Kinetics of Laquinimod Metabolism. Laquinimod was metabolized by human liver microsomes, and at least six primary metabolites, M1 to M6, were detected. A representative HPLC chromatogram is shown in Fig. 1. Identification of M1 to M6 by mass spectrometry (data not shown) demonstrated hydroxylation of laquinimod in the phenyl ring (M1) and in the quinoline moiety (M2-M4). Furthermore, laquinimod was N-dealkylated in the quinoline part of the molecule (M5) and at the aniline nitrogen (M6). The formation of metabolites was NADPH-dependent, linear with time and protein concentration (data not shown). To determine the Michaelis-Menten parameters for the formation of the primary metabolites, laquinimod, at concentrations of 5 to 3000 µM, was incubated for 20 min with microsomes. The apparent K_M, V_max, and CL_int are shown in Table 1 and the saturation curves in Fig. 2. The metabolite formation exhibited, in general, single-enzyme Michaelis-Menten kinetics with K_M in the range 0.09 to 1.9 mM and V_max from 22 to 120 pmol/mg/min for M1 to M6. However, the formation of M4 displayed biphasic kinetics with a second CL term that was not saturated over the substrate concentration range used in this study.

View larger version (20K): [in this window] [in a new window]   FIG. 1. HPLC radiochromatogram of metabolites formed in incubations of laquinimod with human liver microsomes.

View larger version (16K): [in this window] [in a new window]   FIG. 2. Saturation curves for the formation of metabolites (M1-M6) from laquinimod in human liver microsomes.

Hydroxylation in the quinoline moiety was the major pathway, representing 66% of total metabolism, whereas the N-demethylation, N-deethylation and the hydroxylation in the aniline part of the molecule constituted about 19, 10, and 4%, respectively (Fig. 3).

View larger version (16K): [in this window] [in a new window]   FIG. 3. Formation of primary metabolites of laquinimod in human liver microsomal incubations. Heavy arrows denote major metabolites; light arrows represent minor metabolites. , site of metabolism.

Inhibition of Laquinimod Metabolism. Competitive and irreversible inhibitors were used to evaluate the P450 enzymes involved in the metabolism of laquinimod. The formation of metabolites M1 to M6 was quantified and compared with control incubations (without inhibitor) in human liver microsomes. Inhibitors of CYP3A4, ketoconazole and troleandomycin, were found to inhibit the formation of the primary metabolites, whereas no inhibition was seen with any of the other P450 inhibitors used in this study (Fig. 4). In contrast, quinidine was found to stimulate the formation of M2-M3 and M5 to some degree. Furthermore, the formation of metabolite M6 was enhanced in incubations with furafylline.

View larger version (19K): [in this window] [in a new window]   FIG. 4. Effect of the P450 inhibitors furafylline (20 µM), coumarin (25 µM), diclofenac (200 µM), sulfaphenazole (50 µM), quinidine (25 µM), chlorzoxazone (50 µM), ketoconazole (2 µM), and troleandomycin (100 µM) on the formation rate (percentage of control) of laquinimod metabolites M1 to M6, expressed as a sum, at 200 µM laquinimod in human microsomes. Data are the average of two determinations.

View larger version (11K): [in this window] [in a new window]   FIG. 5. Metabolism of laquinimod (100 µM) by cDNA-expressed P450 enzymes.

The Effect of Laquinimod on the Metabolism of Ethinyl Estradiol and Prednisolone. Incubation of ethinyl estradiol (100 µM) with various concentrations of laquinimod within the range 5 to 3000 µM resulted in a decreased metabolism of ethinyl estradiol. An IC₅₀ value for laquinimod of 154 µM for the inhibition of ethinyl estradiol metabolism was calculated. Incubation of prednisolone (100 µM) with various concentrations of laquinimod within the range of 5 to 3000 µM did not affect the metabolism of prednisolone.

The Effect of CYP3A4 Substrates on the Metabolism of Laquinimod. Laquinimod was incubated with known CYP3A4 substrates (ketoconazole, troleandomycin, erythromycin, prednisolone, ethinyl estradiol) at concentrations at which no inhibition to total inhibition occurred.

All the studied compounds were found to inhibit the formation of the laquinimod metabolites, M1 to M6 (Table 4). The strongest inhibition of laquinimod primary metabolism was demonstrated for ketoconazole with an IC₅₀ value of 0.2 µM, followed by troleandomycin, erythromycin, prednisolone, and ethinyl estradiol, with IC₅₀ values of 11, 24, 87, and 235 µM, respectively.

	Discussion

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In the present study, liver microsomal preparations from humans were used to establish the enzymatic basis for the primary metabolism of laquinimod, a new immunomodulator, in humans. Laquinimod was metabolized to the primary hydroxylated metabolites M1 to M4, and dealkylated metabolites M5 and M6. The metabolite formation exhibited single-enzyme Michaelis-Menten kinetics with K_M in the range 0.09 to 1.9 mM and V_max from 22 to 120 pmol/mg/min for M1 to M6. The broad range of K_M values obtained for different metabolic pathways produced by a single enzyme has previously been described for other compounds, for instance, midazolam, which is metabolized by CYP3A4 to metabolites with very different K_M values (Khan et al., 2002).

Hydroxylation at different sites in the quinoline moiety (M2-M4) was the major pathway (66%) followed by N-demethylation (19%), N-deethylation (10%), and hydroxylation in the para position of the phenyl ring (4%). One of the quinoline-hydroxylated metabolites (M2) was also found to be dehalogenated, which might be explained by the fact that some of the rearrangement reactions during P450-catalyzed oxidation of aromatic rings involves migration of the geminal hydrogen atom. This displacement, known as the "NIH shift," may also affect lower halogens such as chloro-substituents (Safe et al., 1976). It remains to be clarified whether any of the laquinimod metabolites display pharmacologic or toxicologic activity. However, metabolites formed by aniline N-dealkylation of structurally related compounds have been shown to enhance cell-mediated immunity in animal models (Eriksoo et al., 1985).

The strong correlation observed between the formation rate of the major metabolites, M2 to M5 (0.9-0.94), M1, M6 (r0.6), and 6ß-hydroxylation of testosterone (CYP3A4) in the panel of human liver microsomes implies that both hydroxylation and demethylation of laquinimod are mediated mainly through enzymes in the CYP3A family.

A good correlation was also obtained toward total P450 content, which is in accordance with the relatively high abundance of CYP3A4 in the human liver (Shimada et al., 1994). Furthermore, a 5-fold variation in metabolism of laquinimod across the panel is also in good agreement with the large variability reported for CYP3A4 activity within a general population (Wilkinson, 1996).

Moreover, the formation of major metabolites was significantly inhibited by ketoconazole and troleandomycin, specific inhibitors of CYP3A4, which further supports the role of CYP3A4 in the metabolism of laquinimod. Interestingly, the formation of some of the metabolites (M2-M3, M5) was stimulated by coincubations with quinidine. Both homotropic and heterotropic cooperativity have been described for CYP3A4 (Ekins et al., 1998; Tang and Stearns, 2001). Quinidine has been found to stimulate (for example) the CYP3A4-catalyzed metabolism of warfarin in human liver microsomes and hepatocytes (Ngui et al., 2001). Quinidine was also found to stimulate the metabolism of diclofenac both in vitro and in vivo in rhesus monkeys (Ngui et al., 2000; Tang and Stearns, 2001). In vivo examples of heterotropic cooperativity are, however, rare, and the importance of a possible stimulation of the laquinimod metabolism in vivo remains to be further studied.

Investigation of secondary metabolism of laquinimod was beyond the scope of the present study. However, preliminary experiments have demonstrated that the N-dealkylated metabolite, M6, is further rapidly metabolized by hydroxylation in the aniline part of the molecule. By using recombinantly expressed enzymes, the reaction was found to be catalyzed by enzymes in the CYP1A and, to a minor extent, the CYP3A family (data not shown). The enhanced level of M6 after coincubations with laquinimod and furafylline, a specific CYP1A inhibitor, also supported the role of CYP1A enzymes in the further metabolism of M6.

Recombinantly expressed CYP3A4 was found to produce the whole pattern of primary metabolites from laquinimod. Furthermore, kinetic parameters obtained with recombinant CYP3A4 demonstrated K_M values of the same order of magnitude as in human microsomes.

However, the formation of the hydroxylated metabolites M3 and M4 was also catalyzed by CYP1A1. CYP1A1 is not constitutively expressed in the liver but is inducible by a variety of compounds (cruciferous, cigarette smoke, polycyclic aromatic hydrocarbons, and dioxins, but also, drug compounds such as antimalarials and the benzamidazols) (Fontaine et al., 1999; Fuhr, 2000), and a minor role for CYP1A1 in the laquinimod metabolism cannot be excluded. The hydroxylated metabolite M3 was also formed in incubations with CYP2C19. Taken together, a very small role by other P450 enzymes in laquinimod metabolism cannot be ruled out completely, but the significance of correlation with CYP3A4 activity and laquinimod metabolism integrated with the inhibition results strongly support a principal role for CYP3A4 in the metabolism of laquinimod. When comparing the metabolism of laquinimod in the present study with that of the chemically related compound, roquinimex (Tuvesson et al., 2000), important similarities are demonstrated. Both compounds are metabolized through a low-affinity process by CYP3A4 to hydroxylated and dealkylated metabolites.

There are a number of drugs that are metabolized preferentially by enzymes in the CYP3A family, and it has been reported that the enzyme may be involved in the metabolism of as much as 50% of drugs used in humans. This includes several classes of drugs, such as calcium channel antagonists, immunosuppressant agents, cholesterol-lowering agents, nonsedating antihistamines, benzodiazepines, and macrolide antibiotics (Shou et al., 1994). Thus, identifying CYP3A4 as a major enzyme in the primary metabolism of a compound may be of clinical importance with regard to drug-drug interactions.

The low affinity between laquinimod and the enzyme (high K_M), demonstrated in the present study will reduce the risk for competitive inhibition of the metabolism of other CYP3A4 substrates. However, possible effects of laquinimod on the metabolism of ethinyl estradiol, the main active component in most oral contraceptives and prednisolone, a glucocorticoid widely used as a complement in MS treatment, were studied in vitro. The mutual inhibition between laquinimod and ethinyl estradiol demonstrated an IC₅₀ value of 154 µM laquinimod inhibition of ethinyl estradiol, whereas no effects were seen on prednisolone metabolism. Preliminary estimates from ongoing clinical studies demonstrate a C_max level at steady state of below 5 µM laquinimod when doses of 0.05 up to 2.4 mg daily were given to healthy volunteers or patients. This level of laquinimod in plasma is far below the calculated IC₅₀ value of 154 µM for inhibiting the ethinyl estradiol metabolism.

In vitro studies were undertaken to study the potential of a number of drugs, known to be CYP3A4 substrates, to influence the metabolism of laquinimod. Incubations were performed to determine IC₅₀ values for the inhibition of the laquinimod metabolism. The strongest inhibition of laquinimod primary metabolism was demonstrated for ketoconazole, with an IC₅₀ value of 0.2 µM, followed by troleandomycin, erythromycin, prednisolone and ethinyl estradiol with IC₅₀ values of 11, 24, 87, and 235 µM, respectively.

Ketoconazole is a relatively specific and potent inhibitor of CYP3A4-mediated metabolism, and the IC₅₀ value in the present study is in good agreement with what has been reported for ketoconazole inhibition of other CYP3A4 substrates (Wang et al., 1999). The IC₅₀ values obtained for troleandomycin and erythromycin in the present study are also in accordance with reported data (Zhao et al., 1999; Echizen et al., 2000).

To predict possible drug-drug interactions in vivo, the in vitro effects of the studied drugs (IC₅₀ value) have to be considered in the light of clinically relevant concentrations. The unbound concentration of inhibitor around the metabolizing enzyme in the liver is one of the key factors determining the extent of drug-drug interactions in vivo. However, for practical reasons, the unbound concentration of drug in plasma is generally considered to correspond to the concentration in the liver and, therefore, is used in predictions of drug-drug interactions in vivo.

Plasma levels of ketoconazole during clinical use generally exceed 1 to 3 µM (Greenblatt et al., 1998). Since the calculated IC₅₀ value for inhibition of laquinimod primary metabolism by ketoconazole is considerably lower than typical clinical plasma concentrations inhibition of laquinimod metabolism is likely to be of clinical importance.

After a single dose of erythromycin (400 mg/kg) a C_max value of 2.5 µg/ml (3.4 µM) was obtained (Kanazawa et al., 2001). Comparing plasma concentration and the IC₅₀ value for erythromycin in the clinically relevant drug-drug interactions of the present study seems unlikely. The macrolide antibiotics there among erythromycin and troleandomycin are mechanism-based inhibitors of CYP3A4 (Yamano et al., 2001). The degree of drug-drug interaction caused by these compounds is considered to depend on the concentration of inhibitor as well as the contact time of inhibitor and enzyme, which may be considered in a clinical situation.

In a recent study, plasma levels of 52.5 ng/l (0.18 nM) and 0.96 µM ethinyl estradiol and prednisolone, respectively, were determined in women taking either oral contraceptives (30 of µg ethinyl estradiol per day) or repeated oral doses of 20 mg of prednisolone per day (Seidegård et al., 2000). Thus, any drug-drug interaction of ethinyl estradiol and prednisolone with laquinimod seems unlikely to occur when the different plasma levels are compared with the IC₅₀ values for the two compounds obtained in the present study.

In conclusion, the present study demonstrates that laquinimod, a novel immunomodulator, is a low-affinity substrate for CYP3A4 in humans. Considering the low affinity between laquinimod and the enzyme, together with plasma concentrations achieved in vivo, the likelihood of an in vivo interaction between laquinimod and other CYP3A4-metabolized drugs in humans might be negated. However, a possible influence of other drugs on the metabolism of laquinimod in humans, which may affect the clearance of the compound, cannot be excluded. Inhibitory effects on the laquinimod primary metabolism of potent and specific inhibitors of CYP3A4, such as ketoconazole, is anticipated and should be considered in the continued clinical program for laquinimod.

	Acknowledgments

 
We thank Lillemor Halvarsson for excellent technical assistance and Örjan Nordle for statistical analysis of the enzyme kinetics.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.104.002238.

ABBREVIATIONS: ABR-215062, laquinimod (N-ethyl-N-phenyl-5-chloro-1,2-dihydroxy-1-methyl-2-oxo-3-quinoline-carboxamide); P450, cytochrome P450; MS, multiple sclerosis; HPLC, high performance liquid chromatography; CL_int, intrinsic clearance.

Address correspondence to: Helén Tuvesson, Active Biotech Research AB, Box 724, SE-220 07 Lund, Sweden. E-mail: helen.tuvesson{at}activebiotech.com

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