Short Communication CYP2C8/9 Mediate Dapsone N-Hydroxylation at Clinical Concentrations of Dapsone

Using selective cytochrome P450 (CYP) inhibitors and clinical concentrations (4 mM) of dapsone (DDS), we found a major contribution of CYP2C9 and little or no contribution (<10%) of CYP3A4 and CYP2E1 to dapsone N-hydroxylation (DDS-NHY) in human liver microsomes. Sulfaphenazole (2.16 mM) and tolbutamide (500 mM), selective inhibitors of CYP2C9 (or 2C8/9), inhibited DDS-NHY by 48 6 14 and 41 6 15%, respectively. The apparent MichaelisMenten Km values for DDS-NHY by cloned CYP2C8, CYP2C9, CYP2C18, and CYP2C19 were 75 mM, 31 mM, 25 mM, and greater than 1 mM, respectively. CYP3A4 and CYP2E1 were incapable of DDS-NHY at 4 mM DDS. S-mephenytoin (360 mM) activated DDS-NHY by human liver microsomes and by CYP2C8 by 43 6 36 and 193 6 16%, respectively. This activation was cytochrome b5-dependent. In contrast, S-mephenytoin inhibited DDS-NHY by CYP2C9, CYP2C18, and CYP2C19 by 27 6 2, 49 6 1, and 32 6 4%, respectively. Because CYP2C18 and CYP19 are expressed at low concentrations in the human liver, these observations indicate that at clinical DDS concentrations, CYP2C9 is a major and CYP2C8 is a likely minor contributor to DDS-NHY in human liver microsomes. Dapsone (DDS) is used to treat Pneumocystis carinii pneumonia in people with AIDS who are intolerant to co-trimoxazole (sulfamethoxazole/trimethoprim). However, the high rate of adverse reactions to DDS in people with AIDS such as leukopenia, thrombocytopenia, methemoglobinemia, and changes in liver transaminase activity often requires discontinuation of therapy with DDS (Safrin et al., 1996). Dapsone N-hydroxylamine (DDS-HA) has been implicated as the causative agent of the adverse reactions to DDS. Dapsone hydroxylation (DDS-NHY) has been shown to be directly related to methemoglobin formation in vivo (Coleman et al., 1992; Mitra et al., 1995; Rhodes et al., 1995). There is evidence to suggest that a combination of oxidative stress and hydroxylamine formation is responsible for the high rate of adverse reactions to arylamine antibiotics in AIDS patients (Carr et al., 1993; Pace and Leaf, 1995; Rieder et al., 1995). DDS-NHY has been reported to be catalyzed by cytochrome P450 (CYP)2C9, CYP3A4, and CYP2E1 (Fleming et al., 1992; Gill et al., 1995; Mitra et al., 1995). Unfortunately, this assignment of CYP isoforms to DDS-NHY is confounded by the use of either DDS concentrations much greater than those encountered in the clinic (Fleming et al., 1992; Gill et al., 1995) or use of an inhibitor concentration [diethyldithiocarbamate (DDC); Mitra et al., 1995], which could lead to inhibition of isoforms other than CYP2E1 (Chang et al., 1994; Ono et al., 1996). The use of disulfiram (CYP2E1 inhibitor) as a metabolic inhibitor in vivo, has been explored as a therapeutic strategy to suppress DDS-NHY; it produced a 65% decrease in DDS-NHY in non-HIV-infected subjects (Mitra et al., 1995). However, in a recent clinical study in HIV-infected patients, fluconazole (a CYP2C and CYP3A4 inhibitor that does not inhibit CYP2E1) unexpectedly inhibited DDS-NHY by 50% (Winter et al., 1998), a magnitude greater than expected based on previously reported in vitro and in vivo data (Mitra et al., 1995). As part of an ongoing series of studies on the prospective in vitro prediction of in vivo drug interactions for drugs used to treat opportunistic infections in AIDS, we have investigated the role of the CYP2C family in mediating DDS-NHY at clinical concentrations (4–8 mM) of DDS. Materials and Methods Chemicals. DDS-HA was synthesized and provided by the National Institutes of Allergy and Infectious Diseases (Bethesda, MD). The purity of the hydroxylamine was determined to be .99% by quantitative thin-layer chromatography. DDS was purchased from Sigma Chemical Co. (St. Louis, MO). S-mephenytoin was a gift from Dr. René H. Levy, Department of Pharmaceutics, University of Washington, Seattle, WA. Human anti-cytochrome b5 and preimmune antibodies were a gift from Dr. Peter Manyike, University of Washington. All other chemicals were of analytical grade and obtained commercially. Human Liver Samples. Livers were procured, processed, and stored as previously described (Rettie et al., 1989). Microsomes from the University of Washington’s human liver bank from human livers numbered 123, 139, 140, 141, and 142, were prepared as previously described (Hickman et al., 1998). Cloned Human Enzymes. All cloned enzymes were obtained from Gentest (Woburn, MA). Unless specified, the clones were human lymphoblast-expressed, containing co-expressed CYP reductase and endogenous cytochrome b5. The CYP2C18 cloned enzyme was baculovirus insect cell-expressed Gentest Supersome preparation lacking coexpressed cytochrome b5. Therefore, CYP2C18 was always mixed with the Gentest Supersome vector insert control This work was supported by NIH AI 27664 from the National Institutes of Health. DDS-HA was synthesized by Starks Associates, Inc. under Contract NO1 A1 050515 from the National Institutes of Health. 1 Abbreviations used are: DDS, dapsone; DDS-NHY, dapsone N-hydroxylation; DDS-HA, dapsone N-hydroxylamine; DDC, diethyldithiocarbamate; HLM, human liver microsomes; CYP, cytochrome P450. Send reprint requests to: Jashvant D. Unadkat, Ph.D., Department of Pharmaceutics, Box 357610 H272 Health Sciences Building, University of Washington, Seattle, WA 98195-7610. E-mail: [email protected]. 0090-9556/00/2808-0865–868$03.00/0 DRUG METABOLISM AND DISPOSITION Vol. 28, No. 8 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. DMD 28:865–868, 2000 /1474/838135 865 at A PE T Jornals on Jne 3, 2017 dm d.aspurnals.org D ow nladed from containing coexpressed cytochrome b5 (1:1, v/v) when incubations containing this enzyme were conducted. Enzyme Assays. The 250-ml incubation matrix consisted of 1 mg/ml microsomal protein, 1 mM EDTA, and 1 mM GSH in 50 mM HEPES buffer, pH 7.4 at 37°C. Concentrated DDS stock solutions were prepared in 0.1 N HCl and diluted in HEPES buffer. All inhibitors were prepared as aqueous solutions unless specified, and appropriate controls were conducted. The final concentration of organic solvent in the incubation mix did not exceed 1% v/v. Reactions were preincubated for 5 min, then 1 mM NADPH was added and the reaction was terminated with 25 ml of 2 N HCL after an additional 5 to 15 min. Samples were left on ice for 10 min, then centrifuged (20,000g) at 4°C for 10 min, and the supernatant was injected onto the HPLC. The hydroxylamine was stable in the incubation matrix containing heat-denatured protein for at least 40 min (0% loss) at 37°C. The hydroxylamine spiked into the full sample matrix and treated with 0.1 N HCl was stable for greater than 3 h (0% loss) at room temperature (;20°C). HPLC Assay. DDS-HA was assayed by a specific validated method using an electrochemical reverse-phase HPLC method with minor modifications (Mitra et al., 1995). Data Analysis. Nonlinear estimation of Vmax, Km, residual effect model IC50, and apparent IC50 was performed using PCnonlin (Scientific Consulting Inc., Apex, NC) and reported as the parameter 6 S.E.M. All statistical data are reported as the mean 6 S.D. of triplicate determinations. The unpaired t-test was used to detect significant differences with P # .05. Results and Discussion CYP-mediated DDS-NHY was found to be biphasic in HL141 microsomes with Km values of 5.1 6 3.3 and 189.9 6 52.5 mM (data not shown), which agrees closely with Mitra and colleagues (Mitra et al., 1995). Because clinical concentrations of DDS therapy are 4 to 8 mM, we conducted all our microsomal incubations at a concentration (4 mM) comparable to the lower Km value. Of the selective P450 inhibitors (Hickman et al., 1998) used, only sulfaphenazole (2.16 mM; CYP2C9) and tolbutamide (500 mM; CYP2C8/9) significantly inhibited (by 48 6 14 and 41 6 15%, respectively) DDS-NHY in human liver microsome (HLM); HL123, 139, 141; Fig. 1A). The inhibition observed reflects CYP2C9 inhibition. It should be noted that tolbutamide at the concentration used was not expected to significantly inhibit CYP2C8, and that sulfaphenazole does not inhibit CYP2C8 (Fig. 1B). In addition, a clinical concentration of fluconazole (20 mM), an inhibitor of CYP2C9, CYP2C19, and CYP3A4 (Fig. 1B) inhibited DDS-NHY in HLM by 41 6 15% whereas 360 mM Smephenytoin activated this reaction by 43 6 37% (Fig. 1A). Lymphoblast-expressed CYP2C8, CYP2C9, CYP2C18, and CYP2C19 were all capable of forming DDS-HA from 6.25 mM DDS at a rate of 0.0357, 0.918, 0.684, and 0.0349 pmol/min/pmol of P450, respectively. In contrast, lymphoblast-expressed CYP3A4 and CYP2E1, and Supersome CYP2E1 with coexpressed cytochrome b5 did not show detectable DDS-NHY at a DDS concentration of 4 mM. Velocity profiles for DDS-NHY with increasing substrate concentration (1.6– 300 mM) were typical hyperbolic Michaelis-Menten profiles for CYP2C8, CYP2C9, and CYP2C18 (Fig. 2A). DDS-NHY by CYP2C19 displayed autoactivation within the clinical range (4–12.5 mM) as demonstrated by the nonlinear Eadie-Hofstee curve (Fig. 2B). The apparent Michaelis-Menten Km values for DDS-NHY by CYP2C8, CYP2C9, CYP2C18, and CYP2C19 were 75 mM, 31 mM, 25 mM, and .1 mM, respectively. To gain insight into the activation of DDS-NHY by S-mephenytoin, we measured DDS-NHY in expressed 2C enzymes. S-mephenytoin (360 mM) inhibited DDS-NHY by CYP2C9, CYP2C18, and CYP2C19 by 27 6 2, 49 6 1, and 32 6 4%, respectively, and activated this reaction by CYP2C8 by 194 6 16% (all P # .05; Fig. 3A). Cytochrome b5 appears to have an important role in the mechanism of the S-mephenytoin-mediated activation. CYP2C8 Supersomes required the addition of cytochrome b5 to show the activation phenomena and the extent of S-mephenytoin activation in HLMs (68% greater than that expected for control DDS-NHY) was inhibited 60% (P # .05) by a validated cytochrome b5 antibody (Lee et al., 1997) at a microsomal protein/antibody ratio of 1:30 (Fig. 3A). The velocity profile for CYP2C8-mediated DDS-NHY in the presence of 360 mM S-mephenytoin was a typical hyperbolic Michaelis-Menten profile (Fig. 3B). However, both the apparent Km and Vmax values were increased 56 and 218% from the control values of 58 mM and 0.440 pmol/min/pmol of P450, respectively, consistent with a substantial cytochrome b5-mediated effect on the catalytic rate constant. To quantify the inhibitory capacity of fluconazole towards DDSNHY, we determined the IC50 of fluconazole. An IC50, rather than a Ki, was determined because multienzyme involvement in DDS-NHY [shown by biphasic Eadie-Hofstee transformations by Mitra and colFIG. 1. Inhibition of DDS-NHY at 4 mM DDS by CYP-selective inhibitors in HLMs (HL123, 139, 141) (A) and Gentest human lymphoblast-expressed 2C8, 2C9*1, 2C19, and baculovirus-insect-cell-expressed (Supersome) 2C18 human CYP enzymes (B). Tolbutamide and S-mephenytoin incubations and their matched controls contained 1% v/v methanol. Values represent the mean percentage of control activity 6 S.D. of triplicate determinations. ND, not detectable. *, significant inhibition (P # .05). TAO, troleandomycin. 866 WINTER ET AL. at A PE T Jornals on Jne 3, 2017 dm d.aspurnals.org D ow nladed from