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Vol. 29, Issue 2, 100-102, February 2001

SHORT COMMUNICATION

Ritonavir, Efavirenz, and Nelfinavir Inhibit CYP2B6 Activity in Vitro: Potential Drug Interactions with Bupropion

	Abstract

Top Abstract Introduction Results and Discussion References

Since antiretroviral drugs are known to inhibit many cytochrome P450 isoforms, the inhibition of CYP2B6 by non-nucleoside reverse transcriptase inhibitors and viral protease inhibitors was studied in vitro in human liver microsomes using bupropion hydroxylation as the CYP2B6 index reaction. Mean IC₅₀ values (µM) for inhibition of bupropion hydroxylation were: nelfinavir (2.5), ritonavir (2.2), and efavirenz (5.5). The reaction was only weakly inhibited by indinavir, saquinavir, amprenavir, delavirdine, and nevirapine. The inhibition of bupropion hydroxylation in vitro by nelfinavir, ritonavir, and efavirenz indicates inhibitory potency versus CYP2B6 and suggests the potential for clinical drug interactions.

	Introduction

Top Abstract Introduction Results and Discussion References

Depression frequently accompanies HIV infection (Fernandez and Levy, 1991; Judd and Mijch, 1996; Lyketsos et al., 1996; Ferrando et al., 1997), and studies estimate that depression affects 32 to 56% of HIV-infected persons (Ferrando et al., 1997). Depression associated with HIV infection may be a result of dealing with a progressive physical disability, discrimination, or loss of a social support system (Chesney and Folkman, 1994; Judd and Mijch, 1996; Lyketsos et al., 1996). Alternatively, depression may be due to the virus itself affecting the central nervous system (Lyketsos et al., 1996).

Several studies have examined the efficacy and tolerability of different antidepressants in treating depression in HIV-infected individuals. Bupropion is an atypical antidepressant that is often administered when other agents have been ineffective (Walker et al., 1993). Bupropion treatment of depression in HIV patients has been described in uncontrolled studies (Fernandez and Levy, 1991; Avants et al., 1998).

Viral protease inhibitors inhibit a number of cytochrome P450 (CYP¹) isoforms in vitro, including CYP3A4 (Eagling et al., 1997; Malaty and Kuper, 1999; von Moltke et al., 1998, 2000). The non-nucleoside reverse transcriptase inhibitors efavirenz and delavirdine inhibit CYP3A4, -2C9, and -2C19 (Voorman et al., 1998; von Moltke et al., 2001). The effect of antiretroviral agents on CYP2B6 activity is not established. CYP2B6 is the primary enzyme mediating the formation of hydroxybupropion from bupropion in human liver microsomes and can be used as a probe for CYP2B6 activity in vitro (Faucette et al., 2000; Hesse et al., 2000). The objective of this study was to investigate the possibility of in vitro inhibition of CYP2B6 by several antiretroviral agents, using bupropion hydroxylation as the index reaction.

Experimental Procedures

Materials. Bupropion hydrochloride and hydroxybupropion were kindly provided by Glaxo-Wellcome (Research Triangle Park, NC) and trazodone was provided by Mead Johnson (Evansville, IN). Antiretroviral agents were provided by their manufacturers or extracted from their clinical dosage forms. Other chemicals and reagents were obtained from commercial sources. Liver samples from donors with no known liver disease were obtained from either the National Disease Research Interchange (Philadelphia, PA) or the Liver Tissue Procurement and Distribution Service (Minneapolis, MN). The microsomes were prepared as previously described (von Moltke et al., 1993).

Incubations Using Human Liver Microsomes. A screen of bupropion hydroxylation inhibition was performed by adding 10 and 100 µM inhibitor to incubation tubes with bupropion (final concentration 50 µM), and solvent was evaporated to dryness before addition of cofactors and protein. Incubations were performed in duplicate as previously described (Hesse et al., 2000). Briefly, incubation mixtures contained 50 mM potassium phosphate buffer (pH 7.5), 0.5 mM NADP⁺, an isocitrate/isocitric dehydrogenase regenerating system, and 5 mM MgCl₂. Reactions were initiated by the addition of microsomal protein (final concentration 0.25 mg ml¹) and terminated after 20 min by the addition of 1 N HCl. Trazodone was used as an internal standard. Incubations with 10 µM inhibitor were performed using four different human livers, and incubations with 100 µM inhibitor were performed using one human liver with high CYP2B6 activity.

To generate IC₅₀ estimates for antiretroviral drugs that significantly inhibited bupropion hydroxylation at 10 µM, varying concentrations (0-50 µM) of these agents (nelfinavir, ritonavir, efavirenz) were added to incubation tubes. Microsomes from four different human livers were used. Incubations were performed as previously described (Hesse et al., 2000).

Concentrations of hydroxybupropion were determined by high-performance liquid chromatography using a method adapted from Cooper et al. (1984) and described by Hesse et al. (2000). Briefly, a 300- × 3.9-mm µBondapak C₁₈ column (Waters Associates, Milford, MA) was used with a flow rate of 2 ml min¹, ultraviolet detection at 214 nm, and a mobile phase of 79% 50 mM KH₂PO₄ (pH 3) and 21% acetonitrile.

Preincubations. To evaluate the effect of preincubation on inhibitory potency, 1 or 10 µM inhibitor (nelfinavir, ritonavir, efavirenz, or delavirdine) was preincubated for 15 min with cofactor, buffer, and microsomes from one human liver with high CYP2B6 activity (von Moltke et al., 2000). As a control, cofactor, buffer, and microsomes were added to a tube containing no inhibitor and were preincubated for 15 min. The reaction was started by addition of an aliquot of the mixture to another tube containing bupropion (50 µM final concentration). The reaction was terminated and processed as described above.

Data Analysis. IC₅₀ values for chemical inhibitors were determined by nonlinear regression analysis of data using a fixed concentration of substrate as previously described (Hesse et al., 2000).

	Results and Discussion

Top Abstract Introduction Results and Discussion References

Inhibitory activity of antiretroviral agents at 10 and 100 µM is shown in Table 1. Structures of bupropion, hydroxybupropion, and agents causing substantial CYP2B6 inhibition are shown (Fig. 1, A and B). Mean IC₅₀ values ± S.E. for nelfinavir, ritonavir and efavirenz versus hydroxybupropion formation from bupropion (50 µM) were 2.5 ± 0.4, 2.2 ± 0.1, and 5.5 ± 0.8 µM, respectively (Fig. 2). Amprenavir (100 µM) was a weak inhibitor of bupropion hydroxylation, inhibiting to 49% of control, whereas other antiretroviral agents at 100 µM produced minimal inhibition of bupropion hydroxylation (Table 1). Preincubation of CYP2B6 with nelfinavir, ritonavir, efavirenz, and delavirdine did not substantially alter the inhibitory potency of these antiretroviral agents. Compared with non-preincubated samples, inhibition of CYP2B6 activity due to nelfinavir (1 µM) and ritonavir was slightly diminished by preincubation, whereas inhibition by efavirenz and delavirdine was slightly enhanced by preincubation (Table 2).

View this table: [in this window] [in a new window]   TABLE 1 Percentage of control activity of bupropion hydroxylation with microsomes incubated with inhibitor (10 and 100 µM)

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Structures of bupropion, hydroxybupropion, and the antiretroviral agents. A, structures of bupropion and hydroxybupropion; B, structures of the antiretroviral agents that substantially inhibited bupropion hydroxylation in vitro, ritonavir, efavirenz, and nelfinavir.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Percentage of control activity versus concentration of inhibitor (nelfinavir, ritonavir, or efavirenz) for CYP2B6 index reaction (bupropion hydroxylation). Nel, nelfinavir; Efav, efavirenz; Rit, ritonavir. Each point is the mean (± S.E.) value for four separate human liver microsomal preparations.

View this table: [in this window] [in a new window]   TABLE 2 Percentage of control activity of bupropion hydroxylation with microsomes preincubated with inhibitor versus microsomes not preincubated

Antiretroviral drugs inhibit the activity of several human cytochromes (Eagling et al., 1997; von Moltke et al., 1998, 2000, 2001), but interactions with CYP2B6 have not been established. Although bupropion hydroxylation is a probe for CYP2B6 activity in vitro (Faucette et al., 2000; Hesse et al., 2000), bupropion may be coadministered with antiretroviral agents to treat depressive disorders accompanying HIV infection. The present study demonstrated that nelfinavir, ritonavir, and efavirenz have low IC₅₀ values for inhibition of bupropion hydroxylation. Although the clinical significance of these IC₅₀ values is not fully established, the IC₅₀ values are below or within the usual ranges of clinical plasma concentrations, indicating the possibility of clinically important CYP2B6 inhibition by nelfinavir, ritonavir, and efavirenz in vivo.

Leah M. Hesse
Lisa L. von Moltke
Richard I. Shader
David J. Greenblatt

Departments of Pharmacology and
Experimental Therapeutics
and of Biochemistry,
Tufts University School of Medicine
and the Division of Clinical Pharmacology,
New England Medical Center,
Boston, Massachusetts

	Footnotes

Received September 18, 2000; accepted November 6, 2000.

This work was supported in part by Grants MH34223, MH01237, MH58435, DA05258, DA13209, and RR00054 from the Department of Health and Human Services.

Send reprint requests to: Dr. David J. Greenblatt, Dept. of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA. E-mail: DJ.Greenblatt{at}tufts.edu

	Abbreviations

Abbreviation used is: CYP, cytochrome P450.

	References

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0090-9556/01/2902-100-102$3.00
DMD, 29:100-102, 2001
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics

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