* Pfizer Global Research and Development, Drug Safety Evaluation, Eastern Point Road, Groton, Connecticut 06340-8014; and
Pfizer Global Research and Development, 5-2 Taketoyo-cho, Chita-gun, Aichi-ken 470-2393, Japan
Received December 15, 2000; accepted April 4, 2001
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ABSTRACT |
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Key Words: 5-Lipoxygenase; leukotrienes; microsomal enzyme induction; knockout mice; cytochrome P-450.
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INTRODUCTION |
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Zileuton is a selective inhibitor of 5-LO (Carter et al., 1991) that is effective in treating asthma (Liu et al., 1996
), and it remains the only marketed drug of its class to be approved for this indication. Zileuton induces the hepatic microsomal mixed-function oxidase (MFO) system of mice, an effect characterized by hepatomegaly and increased drug-metabolizing enzyme activity (Rodrigues and Machinist, 1996
). While there are no reports to our knowledge addressing whether humans given zileuton or animals administered other 5-LO inhibitors are similarly affected, development of a drug within this pharmacological class that lacks the potential to elicit this effect would be preferable. In this regard, while drug-related hepatic microsomal MFO induction in humans is manageable, it can affect the metabolism of concurrently administered therapies and environmental chemicals (Ciummo and Katz, 1995
). In addition, some hepatic microsomal MFO inducers have the potential to produce liver tumors in rodents following chronic administration (Inai et al.1988
; Rossi et al.1977
). While the tumorigenic effect of these agents seems to be restricted to rodents (Hinton and Grasso, 1995
; Stevenson et al., 1995
), it can still complicate the drug development process since the appearance of such lesions warrants an appropriate risk assessment.
CJ-11,802 and ZD2138 are 5-LO inhibitors that were discovered and developed by Pfizer and Zeneca, respectively. The development of CJ-11,802 was suspended in early clinical trials due to unacceptable pharmacokinetics (unpublished data), while ZD2138 was tested for efficacy in both aspirin- (Nasser et al., 1994a) and antigen-induced asthma (Nasser et al., 1994b
) but remains unmarketed. Structurally, CJ-11,802 and zileuton are both hydroxyurea-based molecules, while ZD2138 is a 4-methoxytetrahydropyran derivative (Fig. 1
). We uncovered preliminary evidence, expanded upon within this report, that despite the structural differences between CJ-11,802 and ZD2138, they both induce the hepatic microsomal MFO system of rats. Thus, the previously published report on zileuton (Rodrigues and Machinist, 1996
), in conjunction with our preliminary data on CJ-11,802 and ZD2138, suggested to us that induction may be an intrinsic property of 5-LO inhibitors. Therefore, the present study was designed to more fully characterize induction of the hepatic microsomal MFO system of rats following repeated daily oral administration of either zileuton, CJ-11,802, or ZD2138. Subsequently, 5-LO deficient mice, produced by gene targeting, were treated with CJ-11,802 to assess the potential relationship between altered LT status and induction. Since only male 5-LO knockout mice were used, all studies assessing induction were performed using this gender.
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MATERIALS AND METHODS |
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Animal care.
All procedures were in accordance with NIH guidelines for the care and use of animals. Rats and mice were individually housed in plastic cages in a temperature (70 ± 2°F) and humidity (50 ± 5%) controlled facility with a 12 h light/dark cycle (7:00 A.M. to 7:00 P.M.). Animals were allowed free access to food (Agway PROLAB RMH 3200) and reverse osmosis-purified water.
Purified porcine 5-LO assay.
5-LO was purified from porcine leukocytes according to the procedure of Ueda et al. (1986) using a monoclonal antibody (kindly provided by Professor S. Yamamoto, Tokushima University, Japan) conjugated to an Affi-prep 10 column (Bio-Rad). 5-LO activity was measured with or without one of various concentrations of an inhibitor using a continuous spectrophotometric assay monitoring the increase in conjugated diene formation during incubation of the enzyme with AA, as described by Riendeau et al. (1989), with slight modification. The reaction mixture (total volume of 200 µl) contained 0.2 mM CaCl2, 0.2 mM ATP, 0.025% sodium deoxycholic acid, 25 µM AA (2 µl of a 2.5 mM solution in ethanol), test compound (2 µl of the desired 100-fold concentrated solution in dimethylsulfoxide), 5-LO enzyme, and 50 mM TrisHCl buffer (pH 7.4). The reaction was initiated by adding the enzyme to the mixture in ultra-micro cuvettes (10 mm path length and 2 mm interval width). The mixture was then gently stirred for 5 s with a Pipetman (Gilson), and then the increase in absorbance at 235 nm was monitored at 24°C using a Shimadzu model UV-2100S spectrophotometer. The velocity was determined from a slope (at linear increasing phase) after the initial lag time on a plot of reaction progress curve. Total product formation was determined directly from the maximal absorbance change (plateau level).
Yeast-induced LT formation in rat foot.
The bioassay was performed similarly to that described by Opas et al. (1987). Female Crl:CD® (SD) BR rats (150200 g; Charles River Inc., Japan) were administered either 0.1% methylcellulose vehicle or one of various doses of either CJ-11,802 (0.2 to 60 mg/kg), zileuton (3 to 60 mg/kg) or ZD2138 (0.1 to 30 mg/kg) by oral gavage (10 ml/kg) 1 h prior to the sub-plantar injection of 0.1 ml (20 µl) of a 3% brewer's yeast suspension. Animals were sacrificed by cervical dislocation 0.5 h after the yeast injection to assess the resulting LT formation in the foot. The foot was amputated, frozen in liquid nitrogen and subsequently stored (80°C) until analysis. The frozen foot was crushed, mixed with 5 ml of acetonitrile:water (2:1) extraction solution, pulverized in a Waring® blender, and clarified by centrifugation at 10,000 rpm for 10 min at 4°C. LTs in the supernatant fraction were extracted by a C18 Sep-Pak cartridge (Waters/Millipore) and dried under vacuum. The samples were dissolved in 0.2 ml of acetonitrile:water:triethylamine (30:70:0.1) solution, and the concentration of LTs was quantitated by RIA (New England Nuclear) using LTC4 as a standard. The efficiency (31%) of extraction was determined by an addition of authentic standard.
Animals and treatment regimens used to assess hepatic microsomal MFO activity.
Male Crl:CD® (SD) BR rats (150200 g) were obtained from Charles River, Inc., Kingston, New York, while male 5-LO knockout mice (2030 g) were kindly provided by Dr. Beverly Koller of the University of North Carolina. Inactivation of the 5-LO gene in the knockout mice was accomplished by deleting a 1.25 kb genomic fragment that encodes 35 highly conserved amino acids (Goulet et al., 1994). The original gene-targeting experiment was performed in embryonic stem (ES) cells from murine strain 129 (E14TG2a). Chimeric mice derived from targeted E14 ES cells successfully transmitted the mutant 5-LO allele to their offspring. All genotyping was performed by Southern analysis as described previously (Goulet et al., 1994
). We wished to produce a congenic strain of the 5-LO mutation in the murine strain DBA/1J. The congenic strain was derived by transferring the mutant allele to DBA/1J mice by repeated backcrossing. After 10 generations of backcrosses to the DBA/1J strain, the mutant 5-LO congenic mice are statistically 99.9% of the host strain (Mouse Genome Database, 1996
). The knockout (5-LO -/-) mice used in this study were all N-10 congenic, while the wild-type (5-LO +/+) controls were inbred DBA/1J (2030 g) obtained from Jackson Laboratories (Bar Harbor, ME). The absence of the 5-LO enzyme was confirmed in some randomly selected knockout mice by measuring zymosan-stimulated LT production in the peritoneal cavity as described by Griffiths et al. (1997).
In studies to characterize the effects of 5-LO inhibitors on the hepatic microsomal MFO system, animals (at least 5 males/dose/drug) received CJ-11,802 (0, 10, 50, or 200 mg/kg/day to rats; 0, 50, 100, or 200 mg/kg/day to wild-type mice), zileuton (0, 10, 60, or 300 mg/kg/day to rats) or ZD2138 (0 or 200 mg/kg/day to rats) once daily by oral gavage (10 ml/kg) for 14 (zileuton and ZD2138 to rats; CJ-11,802 to wild-type mice) or 30 (CJ-11,802 to rats) consecutive days, while the corresponding controls were given an equivalent volume of 0.5% methylcellulose vehicle.
Subsequent to the completion of the dose-response study in wild-type mice described above, separate groups of male wild-type and 5-LO knockout mice (7/group) received either CJ-11,802 (200 mg/kg/day) or 0.5% methylcellulose vehicle once daily by oral gavage (10 ml/kg) for 14 consecutive days. In all instances, the dosing regimens of the 5-LO inhibitors used in this study did not produce clinical signs of toxicity or adversely affect the food consumption or body weight gain of any of the animals (data not shown).
Approximately 24 h after their last dose, all mice and rats were killed by pentobarbital overdose after an overnight fast. Immediately after death, livers were rapidly removed, weighed, and sections from some of the animals were chilled in preparation for assessment of MFO activity as outlined below.
In vitro assessment of hepatic microsomal MFO activity.
Microsomes were prepared as described previously (Amacher and Smith, 1987) from all control rats and those given the highest doses of CJ-11,802 (200 mg/kg/day), zileuton (300 mg/kg/day), and ZD2138 (200 mg/kg/day). In addition, microsomes were isolated from the livers of all of the wild-type and 5-LO knockout mice that were administered 0.5% methylcellulose vehicle and CJ-11,802 at a dose of 200 mg/kg/day.
Hepatic microsomal cytochrome P-450 (CYP) content was assayed by the method of Omura and Sato (1964) as modified by Guengerich (1982). NADPH cytochrome c reductase (CRed) activity was assayed as described by Guengerich (1982) using horse heart cytochrome c as a substrate. Total protein was assayed by the BCA method (instructions 23230/23225, Pierce Chemical Co., 1986) at 37°C using bovine serum albumin as a standard. Microsomal CYP content and CRed activity were determined in both mice and rats, while the additional analyses described below were only performed in the latter species.
Ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activities were determined using the methods of Pohl and Fouts (1980) and Lubet et al. (1985), respectively. Incubation mixtures included microsomal protein (about 100 µg), NADPH (125 µM), MgCl2 (25 mM) and either 7-ethoxyresorufin (1.5 µM) or 7-pentoxyresorufin (10 µM), all in 0.05 M Tris buffer (pH 7.4). Reactions were conducted for 5 min (37°C) and terminated with the addition of methanol. In both cases, the production of resorufin was measured fluorometrically (550 nm excitation/585 nm emission). P-nitroanisole O-demethylase (p-NOD) activity was determined using the procedure of Netter and Seidel (1964). Incubation mixtures included microsomal protein (about 0.5 mg), NADP (0.45 mg/ml), glucose-6-phosphate (0.14 mg/ml), glucose-6-phosphate dehydrogenase (20 units), MgCl2 (0.76 mg/ml) and p-nitroanisole (0.125 mM), all in 0.1 M sodium phosphate buffer (pH 7.4). Reactions were conducted for 15 min (37°C) and terminated with the addition of 10% trichloroacetic acid. The production of p-nitrophenol was measured spectrophotometrically (400 nm). All in vitro analyses were conducted using either a Cobas-bio-autoanalyzer from Roche Diagnostic Systems (Nutley, NJ), a Perkin-Elmer LS50-B luminescence spectrometer (Norwalk, CT), or a Beckman DU 650 spectrophotometer (Fullerton, CA).
Leukotriene production ex vivo.
Blood was obtained from wild-type and 5-LO knockout mice at death by cardiac puncture and rapidly heparinized (10 units/ml). LT production in blood was initiated as described by Foster et al. (1990). Briefly, the calcium ionophore A23187 was added in dimethylsulfoxide to a final concentration of 20 µM. The blood was incubated at 37°C for 60 min and plasma obtained by centrifugation. Samples were stored at 20°C until assayed for LTB4 by enzyme immunoassay (Perseptive Diagnostics) according to the kit manufacturer's instructions.
Statistics.
Data collected from the experiment with wild-type and 5-LO knockout mice were analyzed by a 2-way analysis of variance (ANOVA) and linear contrasts to test whether the observed effects were pharmacologically mediated or due to some other mode of action. All other data were analyzed using either Dunnett's or Student's t-test where appropriate. All statistical tests were 2-tailed at the p = 0.05 level of significance.
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RESULTS |
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The effect of 5-LO inhibitors on the hepatic microsomal MFO system of rats.
The spectrum of changes associated with induction (i.e., effects on liver weight and hepatic microsomal drug metabolizing enzyme activity) differed between the 3 compounds. Specifically, the repeated daily oral administration of CJ-11,802 (10, 50, or 200 mg/kg/day) and zileuton (10, 60, or 300 mg/kg/day) to rats caused a dose-related increase in relative liver weight (% liver-to-body weight ratio) compared to corresponding controls, while animals treated with ZD2138 (200 mg/kg/day) were unaffected (Table 1). Relative liver weights were increased in rats given CJ-11,802 at 50 and 200 mg/kg/day by 27 and 157%, respectively, while the corresponding elevations in animals given zileuton at 60 and 300 mg/kg/day were 20 and 54%, respectively.
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DISCUSSION |
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While our initial studies in rats suggested that induction might be related to 5-LO inhibition, subsequent analysis of the data appeared to refute this contention. First, the hydroxyurea-based compounds affected the hepatic microsomal MFO system of rats differently from ZD2138. For example, while all 3 5-LO inhibitors increased the activities of p-NOD and CRed, only the hydroxyurea-based compounds induced EROD and PROD, which are indicators of CYP1A1 (Kedderis et al.1991) and CYP2B (Paolini et al.1995
) metabolism, respectively. In addition, increases in relative liver weight and total hepatic CYP content only occurred in the rats that were given the hydroxyurea-based compounds. Thus, induction following the administration of ZD2138 to rats was less extensive than that elicited by the hydroxyurea-based compounds, despite the former agent being the most potent of the 3 5-LO inhibitors in vivo. Secondly, induction in rats did not parallel the pharmacologic potency of the test agents in vivo. For example, the ED50 values of CJ-11,802 and zileuton for inhibiting yeast-induced LT formation in rats were 4.1 and 33.0 mg/kg, respectively. In the dose-response studies, however, hepatomegaly was first observed in rats given CJ-11,802 and zileuton at dose levels of 50 and 60 mg/kg/day, respectively. In addition, relative liver weights continued to increase up to the highest doses of CJ-11,802 (200 mg/kg/day) and zileuton (300 mg/kg/day) that were tested, despite these doses being well beyond the respective ED50 value for each compound. The induction and efficacy studies were, however, performed at different facilities using opposite sexes, and a direct comparison of such data may not be reliable. In this regard, induction of the hepatic microsomal MFO systems of rats can be influenced by the sex of the animal (Beierschmitt et al.,1984
). Therefore, our studies in rats could not definitively link induction in rats by 5-LO inhibitors to their primary pharmacological activity.
In recent years, knockout mice have been used extensively in many areas of pharmaceutical and academic research (Ryffel, 1997). 5-LO knockout mice have been recently developed and used to elucidate the role of LTs in the inflammatory process (Chen et al., 1994
; Goulet et al., 1994
), collagen-induced arthritis (Griffiths et al., 1997
) and pulmonary hypertension in hypoxic rats (Voelkel et al., 1996
). Drawing and expanding upon these latter reports, we used wild-type and 5-LO knockout mice to more definitively ascertain the relationship, if any, that altered LT status has on the induction phenomenon associated with the administration of 5-LO inhibitors.
In our study with wild-type and 5-LO knockout mice, separate groups of animals were administered either CJ-11,802 (200 mg/kg/day) or 0.5% methylcellulose vehicle once daily for 14 consecutive days. This dosing regimen caused induction in rats, and produced no untoward effects in the preliminary dose-escalation study in wild-type mice. With this design, if induction were related to altered LT status, it should have occurred to a similar extent in the knockout control, knockout treated, and wild-type treated animals compared to the wild-type controls. If, however, induction were not related to LT status, both the wild-type and knockout treated groups would respond similarly to the induction stimulus compared to corresponding controls. In this mouse study, comparable induction occurred in animals given CJ-11,802, regardless of whether or not they expressed the 5-LO enzyme. Therefore, we conclude that induction of the hepatic microsomal MFO system by CJ-11,802, and by inference other 5-LO inhibitors, is not related to altered LT status.
While the mechanism of the induction reported here is unknown, the metal chelating properties of the test agents could be a contributing factor. In this regard, hydroxyureas are chelators of iron (Carter et al., 1991; McMillan and Walker, 1992
), and the hepatic microsomal MFO system of rats (Becking, 1972
, 1976
) and mice (Catz et al., 1970
) is induced following the administration of an iron-deficient diet. In addition, our study indicates that induction in rats following the administration of ZD2138, which does not chelate iron (McMillan and Walker, 1992
), was minimal compared to that observed with CJ-11,802 and zileuton. Despite these facts, however, it is unlikely that the iron-chelating properties of the hydroxyureas are solely responsible for the induction that they cause. In this regard, we report that CJ-11,802 and zileuton caused pronounced increases in the hepatic CYP content of rats, while induction following the administration of an iron-deficient diet is not associated with this change (Becking, 1976
). The dynamics of iron deficiency, however, are likely to be different when the condition is elicited chronically through manipulation of the food versus following the intermittent administration of a chelator to an animal fed a nutritious diet ad libitum. Recently, we demonstrated that CJ-11,802 and zileuton induce the microsomal MFO enzymes of rat liver slices in vitro (Chi et al., 1999
). This system was developed to be used, in conjunction with in vivo studies, to better understand the mechanism of induction elicited by these agents, including the possible involvement of altered iron status.
In conclusion, CJ-11,802, zileuton, and, to a lesser extent, ZD2138 induce the hepatic microsomal MFO system of rats. While the precise mechanism of this effect is not known, it is unrelated to altered LT status. Thus, our data with 5-LO knockout mice indicates that it is possible to discover 5-LO inhibitors that lack the potential to elicit this undesirable effect on the liver.
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ACKNOWLEDGMENTS |
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NOTES |
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This work was presented in part at the annual meeting of the Society of Toxicology, Seattle, WA, 1998.
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